By definition a monoclonal antibody is of one Ig subtype, while a polyclonal antibody contains multiple Ig subtypes.  So look at our product isotype description and if it says: IgG1, IgG2a, IgG2b, IgM, etc, these will be monoclonal, whereas: Goat IgG, Rabbit Ig, etc. will be polyclonal. Most of our polyclonal antibodies also have the term poly in the clone name.

Perform a search for your species using our search box at the top of the web page. Alternatively, use our advanced search drop down menu for Species Reactivity and select your choice of species.  For a broad overview of all our cross-reactive antibodies, use our Antibody Cross-reactivity Chart.

On each product data sheet there is a link to all of the available MSDS.  Or check our website under "Support". If your product isn't covered in this section, please contact BioLegend Technical Services.

100% Satisfaction Guarantee. If BioLegend's product does not perform as described on its product data sheet, we'll replace it or refund 100% of the original purchase price

There is an acceptable tolerance range between 2°C and 8°C, for all products recommended to be stored at 4°C.  If your refrigerator at any time has temperatures outside of this range, we recommend you adjust its settings or use a different refrigerator.  

BioLegend does not epitope map antibodies. Sometimes this information is indicated on the datasheets, if published, but generally, we will not know the exact epitope of the antibody.

Most of our products (unless stated otherwise) have a guaranteed shelf life until their expiration date, when under proper storage and handling conditions as instructed on our Product Data Sheets.  For recombinant proteins, the minimum guaranteed shelf-life is 6 months from the date of receipt by the end-user.

Most products are shipped at ambient temperature.  Our products are sufficiently stable to maintain optimal performance after overnight shipping.

Check the Product Data Sheets on-line to review applications/characteristics for each clone. If you still have questions, please contact BioLegend Technical Services at:

• Toll Free Phone (U.S., Canada): 1-877-BIOLEGEND (246-5343)
  Phone: 858-455-9588
  Fax: 858-455-9587
• E-mail: Click Here

Antibodies offered per test have been pre-titrated for optimal performance in flow cytometry assays. Antibodies offered per plate have been pre-titrated in ELISA. Cell Biology antibodies offered per µl have been pre-titrated for Western Blot. The antibody packed in an μg format (untitrated) is not necessarily always from the same batch number as a test format (pre-titrated) though the same procedures are performed in the quality control testing. Please also note that not all test formats of the antibodies have lower concentrations than the μg format. It depends on a particular batch tested. If the optimal concentration were determined at 1 μg/test for a particular batch, then it would be 100 μg in the 100 tests. The advantage to using a pre-titrated format is that it provides more convenience for the optimal performance in areas such as flow cytometry assays, especially for beginners who may save time and reagents on titration of the antibody.

For U.S. orders, if products are in-stock and your order was made before the shipping cutoff time (contact cs@biolegend.com for more info), they are shipped out that day for you to receive the next day. We typically do not ship on Fridays as to avoid packages sitting unattended over the weekend. International direct orders are shipped on Mondays and Fridays, and typically arrive within 2 - 10 days, depending on how long they are in customs.

For direct orders shipped outside the U.S., shipping costs typically start at USD $50 for antibody orders and $100 for dry ice shipment. This could vary country to country. Additionally, prepayment is required for direct international shipments. Also, taxes and duties will be paid by the recipient upon shipment delivery. In some countries, the customer needs to apply for an import permit. When ordering from a distributor, the payment terms are more flexible, the customer and technical support is more immediate, and the customer has greater convenience in not having to deal with customs agencies, duties/taxes, and currency exchange.

According to a study (Le Roy C, et al. 2009. Cytometry A. 75:882), APC tandems are degraded through a cell-dependent mechanism.

If you swap the filter then it may be best that you test the settings with multipeak (6-8) beads such as our Rainbow Calibration Particles and look at their spread with the original filter and recheck the bead spread with the changed filter.

We have not tested the Maxpar^® Ready antibodies formulated in solution containing EDTA for flow cytometry staining. While it is likely that this will work in majority of the situations, it is best to use the non-EDTA formulated version of the same clone for flow cytometry testing. The presence of EDTA in some situations might negatively affect staining.

BioLegend does not test for pathogens in-house aside from the GoInVivo™ product line. However, upon request, this can be tested on a custom basis with an outside, independent laboratory.

We don't perform epitope mapping in-house. We perform our quality control (QC) test with either surface staining or intracellular staining, as indicated in each product datasheet. When surface staining is performed, it means the antibody recognizes an extracellular epitope. In contrast, if the QC is done with intracellular staining, it means that the protein is expressed in this cellular compartment, but it does not necessarily mean that the epitope recognized is exclusively intracellular. The antibody may still be able to detect the protein if it is expressed or exported to the surface of the cell. It is best to further consult the literature related to the particular clone in this matter.

There are several different methods that can be used to conjugate fluorophores to antibodies. This can depend on the antibody and type of fluorophore being used. One common method is to conjugate the organic fluorophore via primary amines (lysines) on the antibody. Another method conjugates maleimide-labeled fluorophore with antibody via sulfhydryl groups in the hinge region.

Be sure to use the buffers recommended in the protocol that you are using.  BioLegend provides a number of flow cytometry protocols, as well as a page to guide you in selecting the appropriate buffers for your flow cytometry experiments.

Refer to your instrument manual to determine the specifications of your instrument. You may also be able to find specifications in the software provided with your instrument. Note that many instruments are custom built and may not match the standard manual. Also, many instruments have adjustable filter sets, allowing you to configure your instrument to suitably run many different combinations of fluorophores. Always verify that the instrument you plan to use is capable of detecting the fluorophores in your experimental panel.

Fixation with paraformaldyde generally tends to decrease the fluorescence intensity of bound antibodies, particularly with prolonged exposure. This is especially true for nanocrystals and tandem dyes such as those derived from PE and APC. Excessive fixation can also alter the conformation of proteins, causing loss of antibody reactivity to proteins as well.

The Maxpar^® Ready format antibody clones are formulated in Phosphate-buffered solution, pH 7.2, containing 0.09% sodium azide and EDTA. The regular purified format clones are formulated in solution that does not contain any EDTA. Both formulations are however without any extra carrier proteins.

Isotype controls are recommended for every flow experiment. Isotype controls that do not recognize any known proteins will provide you with information on the background staining of an antibody due to its isotype. For multicolor experiments, fluorescence-minus-one (FMO) controls can help you determine the fluorescence spillover from all your other antibodies and can be used to help in gating positive and negative boundaries. Unstained cells can also provide you with a relative measure of the autofluorescence associated with any particular cell type.

Tandem dyes are fluorophores composed of two distinct fluorophores conjugated together. The resulting tandem uses the excitation property of the donor fluorophore and emission property of the acceptor fluorophore, based on the principles of fluorescence resonance energy transfer (FRET). This allows for novel fluorophores with high stokes shifts (separation between excitation and emission range).

Fluorescence to Protein (f:p) ratio tells you the average number of fluorophore molecules that are conjugated to each antibody for any given lot of product. These values can vary widely between manufacturers and even among different lots from the same manufacturer. When using isotype controls, it is important to verify that your control has a similar f:p as that of the primary antibody.

Compensation is the process of removing spillover (spectral overlap) from other fluorophores into the detector for your fluorophore of choice. For example, due to the wide emission range of FITC, some of the FITC signal “bleeds” into PE giving you false signal in the PE channel. On newer digital instruments, compensation can be applied automatically using single stain controls.

Fluorescence is the emission of light by a substance that has absorbed light. In most cases, emitted light has a longer wavelength, and therefore lower energy, than the absorbed wavelength. In flow cytometry, fluorophores are used to tag antibodies in order to provide a signal upon detection of an antigen on the sample when exposed to a single wavelength laser.

Since tandems can exhibit variation between sources and lots, it is best to use the exact same antibody as a compensation control. It may be best to use compensation beads for setting up the compensation.

If the product web sheet for that clone does not reveal this information then it is possible that we have not tested the fixation compatibility of that particular clone. Clone specific literature search is advisable in such situations. Visit our webpage to learn more.

The larger holes created by the nuclear permeabilization required for FOXP3 may allow cytokines to leak out of the cell, making it harder to detect lowly-expressed cytokines. You may have to use a control where the cells are only permeabilized through the cell membrane.

It should not be frozen as it will lead to loss of biological activity due to dimerization.

We do not recommend freezing our antibodies as this can denature the antibody or cause fluorochromes to uncouple from the antibody during freeze/thaw. In addition, the antibodies can clump and form aggregates, allowing it to bind nonspecifically to cells. We recommend keeping our flow antibodies at 4°C, protected from light.

It is not recommended. It is best to use PBMCs for this testing.

In general our antibodies can be used for cell sorting, but we do not routinely test this in house.  Please note the fluorochrome conjugates are not endotoxin tested and contain 0.09% azide. Typically this concentration is low, so the azide amount will not affect viability of the cells.  As for activation of cells, typically the incubation time with the antibodies for staining is short (~20minutes) and this should not cause activation.  Incubation of antibodies with cells at 4°C or on ice will help prevent activation.

Yes, you can use less cells or less volume per test, as long as the antibody concentration does not change.  For example, if you decrease the staining volume from 100 to 50 µl, you could use half the amount of antibody.  But, if you decrease your cell numbers from 1 million to 100,000 cells, and your staining volume is still 100 µl, then you should still use the data sheet recommended amount of antibody.

It is best to follow protocol as described on the product data sheet. Moreover, RPMI 1640 has a relatively high concentration of phosphate and low calcium ion concentration, which negatively impacts Annexin binding to its target phosphatidylserine (PS). Measurement of cell death by using Annexin V may also be significantly affected by time of incubation on ice, calcium concentration, and type of medium.

It is recommend to perform a titration because sometimes you find you can use less antibody for your own experimental needs, and also to have a feel for how the antibody behaves in your own hands in your particular experiment.

Please contact our sales department.

Due to continuous recycling of many chemokine receptors, it may be worthwhile to consider staining at room temperature or at 37°C if the staining at lower temperature (which can potentially reduce receptor turnover) is not optimal.

According to a study (Asselin-Labat, ML et al. 2007. Nat. Cell Biol. 9:201) expression levels of CD61 may vary with age as well as pregnancy stages. It may be best to test CD61 on 4-8 week old mice.

We don't test our antibodies for in vivo or in vitro applications unless otherwise indicated. Depending on the product, the TDS may describe literature supporting usage of a particular product for bioassay. It may be best to further consult the literature to find clone specific information.

We calculate the concentration by reading the absorbance at 280 nm on a spectrophotometer. The concentration is calculated using the OD280*Dilution Factor/Extinction Coefficient. The extinction coefficient for an antibody with an IgG isotype is 1.4.

Generally, we test  4 to 6 dilutions with the most commonly used target cells (if they are available) for the titration curve, then determine the optimal concentration based on the S/N (Signal/Noise) ratio.

Annexin-Phosphatidylserine binding is lost below pH 5.2 and with prolonged incubation over a temperature of 42°C.

If you are not looking for a specific clone check we recommend to use the most popular clone based on Pubmed or Google Scholar.  This will give you an idea on how often a clone is used compared to another.

Please see our stimulation guide.

Yes

This non specific binding should be kept in mind. You can try testing the antibody first to see how much non specific binding you see in your sample. It is also best to follow routine steps such as Fc blocking and titration of the antibody in order to reduce background while doing flow cytometric staining. You can also consider our True-Stain Monocyte Blocker™ to see if it improves staining (Cat. No. 426101).

Be sure that the isotype control matches the same concentration/amount being used for the primary antibody. Concentrations between isotype and primary antibodies are not always identical. Therefore, using the same volume may not produce equal amounts of antibody.

The test size products are pre-titrated for optimal staining of 1 million cells in 100 µl volume.  On the other hand, µg size products are at a defined concentration regardless of the optimal usage.  It is still really important, regardless of format, to use the correct isotype control at the same amount (µg) as their antibody of interest.  If the concentration of the antibody is not on the vial, then call us or email (by clicking on “More Info” below) to obtain the concentration for your lot of product.

For clone 1412: This clone specifically recognizes cytoplasmic domain of CD20 and thus can only be used for intracellular flow cytometry. In this instance you will need to include the fixation and permeabilization steps. Please follow the intracellular flow cytometry staining protocol.

For clone 2H7: This clone is ok for regular surface staining for CD20 and there is no need for any fixation and permeabilization steps.

Our technical protocols can be found here.

FC is for cell surface staining and ICFC is for intracellular staining.  Yes, the concentrations can be different. The IC antibodies and isotypes have optimized fluorochrome conjugation properties to give optimal performance for IC staining.

All PE, APC and their tandem dyes labeled antibodies have a F/P of 1:1.  Other formats such as FITC or Alexa dyes have various F/P ratios, typically within range of 3-7

It is lot-specific. On average it ranges between 2-4.

Most failures of IL-17 intracellular staining are due to stimulation problems. PMA/Ionomycin in liquid form is very unstable.  We recommend storing single use aliquots at -20°C degrees. The aliquots should be thawed at ambient temperature and diluted in PBS to a working concentration immediately before stimulation.

We don't test this in house. Typically the molecular weight of an IgG antibody is approximately 150kd.

Please refer to our Cell Marker webpage . For a more streamlined approach, check out the Essential Markers webpage. It is also advisable to consult the literature to find out further information for a particular cell type.

For detecting human FOXP3, both 259D and 206D clones give comparable staining based on in-house testing. Please see the Treg homepage for the differences between clone 206D or 259D. 206D recognizes human FOXP3 epitope in the region of amino acids 105-235. The 259D antibody recognizes human FOXP3 epitope in the region of amino acids 105-235. For other mammalian species, 150D is recommended. Learn more with our Treg webpage.

We recommend using the standard 450/50 filter to detect Brilliant Violet 421™, which is the same filter used to detect Pacific Blue™.  We recommend using the 510/50 filter to detect Brilliant Violet 510™. For Brilliant Violet 570™ we recommend the 585/42 bandpass filter, commonly used for Pacific Orange™. For BV605™ detection, we recommend the standard Qdot® 605 filter, 610/20 with a 595LP dichroic. For BV650™ detection, the standard Qdot® 650 filter, 660/20 with a 630LP dichroic, is recommended. For BV711™ detection, we suggest the standard Qdot® 700 filter, 710/50 with a 685LP dichroic. For BV785™ detection, we suggest the standard Qdot® 800 filter, 780/60 with a 750LP dichroic. See the Fluorescence Spectra Viewer for complete excitation and emission data.

Yes, Brilliant Violet 421™ is exceptionally bright and photostable, making it an excellent choice for microscopy. View the data.

Brilliant Violet 421™ antibody conjugates give an exceptional signal-to-noise ratio, in some cases greater than 10-fold better than Pacific Blue™.  It gives PE-equivalent brightness or better on the violet laser.  Brilliant Violet 570™ is a non-tandem polymer, and provides improvements over Pacific Orange™, AmCyan, and Horizon™ V500. Brilliant Violet 570™ antibody conjugates also provide much improvement over Pacific Orange™, by as much as 6-fold.  Brilliant Violet 605™ and Brilliant Violet 650™ are significantly brighter than eFluor® 605 and eFluor® 650, respectively, by as much as ten-fold, but are not much brighter than Qdot® 605. Brilliant Violet 711™ is significantly brighter than eFluor® 700 and eFluor® 650, by as much as ten-fold, but is not much brighter than Qdot® 705. Brilliant Violet 785™ is similar in brightness to Qdot® 800. Since BV605™, BV650™, BV711™, and BV785™ are non-nanocrystals, they are likely to perform better for intracellular staining and are not affected by fixation.  Learn more…

Brilliant Violet™ is a family of highly fluorescent polymers, excitable by the 405 nm violet laser, created by Sirigen based on Nobel Prize winning chemistry. There are seven fluorophores available: BV421™, BV510™, BV570™, BV605™, BV650™, BV711™, and BV785™. Each has a unique emission spectra as well as a unique set of advantages over other 405 nm excitable fluorophores on the market. Brilliant Violet™ fluorophores are suitable for surface or intracellular flow cytometry, providing excellent signal with very little background. For excitation/emission and beta testing data, recommended filters, and comparable flourophores, as well as guidance on the strengths of each member in the family and incorporating them into multicolor panels, see our dedicated Brilliant Violet™ page.

Contact our sales team for more detailed information regarding your lab’s discounts.

Yes, our ordering system will be able to process orders using the old catalog number as well as the new catalog numbers. However, the old catalog numbers will be phased out, so you are encouraged to transition to the new catalog numbers, especially where orders are generated automatically from your systems.

No, BioLegend does not test Ultra-LEAF™ antibodies by functional assays unless otherwise indicated. Due to the possible complexities and variations of uses of biofunctional antibodies in different assays and because of the large product portfolio, BioLegend does not currently perform functional assays as a routine QC for the antibodies. However, we do provide references in which the antibodies were used for functional assays and we do perform QC to verify the specificity and quality of the antibody based on our strict specification criteria.

BioLegend antibodies and reagents that carry the IVD or ASR designation are manufactured in a registered GMP lab, while our other products are manufactured under ISO 13485 certification. Both environments provide high quality products. To learn more about our quality standards visit our Quality Control page at: https://www.biolegend.com/QC.

No, BioLegend does not test for pathogens in-house unless otherwise indicated.  However, we can recommend an outside vendor to perform this testing as needed.

BioLegend's Ultra-LEAF™ (Ultra-Low Endotoxin, Azide-Free) purified antibodies are specifically designed for functional analyses, providing the most accurate results with minimal negative effects. Ultra-LEAF™ antibodies have an endotoxin level < 10 EU/mg, and GoInVivo™ antibodies are pathogen tested and have endotoxin levels < 1 EU/mg of antibody.

Yes, you can since kappa and lambda represent light chains which don't contribute to the background staining.

Refer to the for listed isotype and format of each primary antibody and match the isotype control appropriately.  For example, if you have a PE conjugated primary antibody with Rat IgG1, k isotype, use PE Rat IgG1, k isotype control Catalog # 400407 or 400408, as your isotype control. For your conveniece, most products have their isotype control listed under their "related products".

Your  vial has the antibody concentration on the label, in some cases.  If not, contact tech support or use our lookup tool for the concentration of your lot of antibody.   Match your isotype to the concentration of the primary antibody.

Yes. Please contact techserv@biolegend.com and provide lot information of the kit and its components.

It is best to follow the recommended protocol without unnecessary delays. However if you wish to delay then it may be better to delay the procedure at the blocking step (after coating with the capture antibody). You can add 200 μl of blocking buffer in the wells and either the plates can be kept overnight at 4°C (minimal or no loss of signal) or frozen at -20°C for few days (it may have some impact on the signal). Delaying the procedure at the first step (coating with capture antibody beyond 16-20 hrs at 4°C) is not advisable because it may lead to high background.

It is possible. Generally, coating for 16-20 hours at 4°C is recommended. Longer incubation time may increase the amount of capture antibody bound to the plates and this may also increase the background noise.

We don’t recommend this. Sensitivity of a kit depends on the individual components and their collective validation as a kit and it will not change by adding extra points to the standard curve.

While this may be possible, you may end up with a plateau in signal at higher concentrations of the standard. It is generally recommended to use the concentration range recommended.

This is not recommended. The ELISA MAX™ reagents are optimized for a particular set lot, and they are not recommended for applications other than ELISA.

Antibodies used are different in different kits. The specificity of the antibodies partially dictate how much signal is being detected. Recombinant standards used are different.  First of all, different kits may use recombinant proteins expressed and purified using different method. Second, recombinant proteins expressed from E. coli from the same source can show greater than 10 fold difference in term of immunoreactivity from lot to lot, primarily due to refolding inconsistency. Third, different kit standards can be produced and calibrated against different references. So far there is no universally accepted standardization for cytokine immunoreactivities.
Each BioLegend ELISA product was developed and validated with reagent concentrations and protocols optimized for best analytical robustness.  Any changes to the reagents (standards, antibodies, matching matrices) and protocols etc all affect the final assay performance.

Since the antibodies are validated in house for ELISA, it is empirical to find out if the antibodies work for applications such as flow cytometry. We recommend you use flow cytometry validated antibodies for this purpose.

No. It is not recommended because the ELISA protein standards are not sterile, may contain other carrier proteins, and not tested for bioactivity. Therefore this material is not bioassay grade.

No. Tissue Culture grade plates are designed for cell culture purpose and do not typically have high binding capacity. We recommend using high protein binding plates such as Nunc Maxisorp™ plates (Cat# 423501).

Almost all of BioLegend’s ELISA products can be used for tissue/ cell extract/homogenate samples as long as the samples are prepared in such a way that they are compatible with immune-reactivity:

• Tissues/cells should be lysed or homogenized in a neutral pH buffer that contains no denaturing chemicals (such as urea, thiourea, SDS).

• No or minimal levels of detergent (SDS, Triton X-100 etc).

• No excessive ionic strength (salt concentration greater than physiological ionic strength).

• The buffers should contain sufficient protease inhibitor cocktails to preserve the target proteins from proteolytic degradation by enzymes released from cells.

Theoretically you can but we don't recommend it and we also can't guarantee the performance of the kit as indicated in the product manual. Antibodies may not recognize or show poor binding towards the protein standard if the immunogen protein used to generate these antibodies is different in terms of structure or sequence. It is therefore empirical to find out if it works. It is best to acquire the ELISA kit components from one source.

It is recommended that for accurate results samples be stored aliquoted for one-time use only. However it is empirical to find out if reusing samples work for a particular analyte as it will depend on sample stability.

No, ELISA MAX™ Deluxe sets no longer come with plates, but Coating Buffer and Assay Diluent (for blocking and dilutions) are included in the ELISA MAX™ Deluxe Sets. Plates can be ordered separately (Cat. No. 423501), and instructions on coating the plates are in the sets’ product manuals.

The antibodies in our IL-1b kits were generated against the mature form. However, because the pro form of the protein contains the mature form the antibodies should detect both forms.

No.

Dilution with assay buffer is required to minimize the matrix difference between the samples and the standards to achieve better accuracy.

• Increase incubation times (1st incubation, detection, avidin-HRP or TMB substrate)
• Shake plates during incubation steps
• Make sure that the standard is completely reconstituted before use.
• Increased washing and soaking in between washings to further decrease background.
• If possible read the plate at 570-590 nm for background subtraction.
• Improve duplicate CV% by controlling pipetting error, washing with bigger volume of washing buffer, etc
• Use a 5-PL or 4-PL curve-fitting method for better calculation at the lower end of the curve. This is usually done with a better curve-fitting software, rather than the linear curve fitting.

It depends on how your samples are analyzed whether in duplicate or triplicate. For example you can run 80 samples with no replicates or 40 samples in duplicates and so on.

If coated plates cannot be used immediately, they should be sealed and stored overnight at 4°C.

If you are using the same clone for both the detection and capture antibodies, the epitope may already be occupied by one of the antibodies and prevent binding of the other. You should always choose different clones for your capture and detection antibodies.

The Wash buffer is the same for all the current LEGEND MAX™ kits. All the part numbers on the Wash Buffer bottles in these kits should be the same. For ELISA MAX™ Deluxe and ELISA MAX™ Standard sets, we provide a recipe for the wash buffer on each kit’s technical data sheet. This recipe is the same for all ELISA MAX™ sets.

You can order more assay diluent Cat# 421203.

We typically use a stabilizer for pre-coated plates. The washings were designed to remove these components before you start the assay. If you do not do the washings, the effect on assay performance is negligible.

For our LEGEND MAX™ and ELISA MAX™ Deluxe formats we recommend following the prescribed protocol. We can’t guarantee kit performance once fixed protocols are changed. Below are just general suggestions for our ELISA MAX™ Standard format.

a) Control background noise: For example, increase number of washings and soaking times in between washes.
b) Control assay precision: For example, be more careful and more consistent in your pipetting, use fresh paper towels for tapping plates to avoid contamination of avidin-HRP from dirty paper towels and increase washing volumes.
c) Increase incubation times: However this usually also increases background so the assay sensitivity may not necessarily increase.
d) Concentrate your sample if possible.
e) Use a five parameter logistic curve fitting method, which can accurately calculate sample concentration at the lower end of the standard curve. In many cases, samples indeed contain very low to non-detectable levels. No matter how you manipulate the assay you may still not be able to obtain detectable concentrations.

This is dependent on the targets being detected and the biological processes. Customers are advised to study the difference between serum and plasma for the targets of interest and decide on the sample type to be used for quantification. Depending on targets, there may be a difference in concentrations of the targets between serum and plasma. The most important factor in preparing plasma or serum samples is consistency in preparation to ensure precise measurements. In general, plasma/serum samples should be free of particulate matters, contain no excess lipids, and have no hemolysis.
These types of contaminants will contribute to background, and adversely affect the precision of the assay. The key is to prepare the sample the same way each time. That is, centrifuging samples at the same speed, for the same time, removing the serum or plasma immediately after centrifugation and aliquoting and freezing the samples in the same time. Avoid repeated freezing and thawing cycles.

No, we don’t recommend it.

That information is proprietary. However the clonality (polyclonal or monoclonal) and host species details may be provided upon request.

LEGEND MAX™ kits with Precoated plates:

• Fully validated KIT
• Most convenient, analytically and biologically validation provided, shortest protocol
• Ready to use reagents, including Pre-coated plates

ELISA MAX™ Deluxe:

• Cost effective, includes some buffers, plates not included
• Pre-titrated antibodies, AV-HRP, Recombinant Standard, Coating Buffer, Assay Diluent, and TMB Substrate Reagent.

ELISA MAX™ Standard:

• Cost effective development SET
• Most cost effective, plates not included
• Pre-titrated antibodies, AV-HRP, and Recombinant Standard.
• Optimization required

TMB Substrate Reagent Set (Cat# 421101) contains two components, which should be mixed immediately prior to use. The TMB High Sensitivity Substrate Solution (Cat# 421501) contains everything in one solution with special stabilizers and is a high kinetic substrate solution which generally gives higher signal. The type of substrate used for an assay can affect the optical density (OD) achievable and the time required to achieve the desired OD.

Since every sample is unique, it is difficult to predict as this may depend on the sample preparation and the nature of the analyte. For LEGEND MAX™ kits refer to the respective ELISA manual for more information.

For ELISA MAX™ Standard format the sensitivity should match the ELISA MAX™ Deluxe version if BioLegend components are used. As for individual LEGEND MAX™ and ELISA MAX™ Deluxe formats the sensitivity values are mentioned in the manual for each kit.

BioLegend's LEGEND MAX™ Kits are guaranteed for 3 months from the date of receipt. ELISA MAX™ sets are guaranteed for 12 months from the date of receipt. For lot-specific expiration date, refer to the box label on each Kit or Set.

This may be dependent on the cytokine being detected, but in general scientists use PBS, pH7.4 or carbonate buffer at pH 9.5.

LEGEND MAX™ Kits are ready-to-go, designed for ease-of-use, minimizing requirements for coating the ELISA plates and preparing buffer dilutions.  We recommend LEGEND MAX™ Kits for users who are new to ELISAs or are short on time.

http://www.biolegend.com/media_assets/support_resource/elisa_troubleshooting.pdf

Currently, it is very difficult to  obtain exactly the same  sample concentrations at pg/ml levels when comparing  different cytokine kits from different vendors, partly because of following reasons:
• The immunoreactivity for the reagents vary from different suppliers.
• In the area of cytokine quantification particularly for those cytokines at pg/mL ranges, the general consensus is that it is more important to have matching biological trend instead of matching absolute concentrations.  This is to say that the same cytokine measured by different kits from different vendors should show the same pattern of biological changes predicted for the relevant biological treatments.
•  For some cytokines, it is not uncommon  that  the cytokine concentrations vary a few-fold between kits for the reasons mentioned above.
• It is critical that kits produced by the same vendor  to keep its assay products consistent from lot-to- lot over time. We tested  ELISA standards from competitor's kits. Results showed clear discrepancies in the relative potency of the standards in term their immunoreactivities.

Azide is an irreversible inhibitor of HRP.

The samples can be kept overnight at 4°C while being protected from exposure to light and be read the next day. There may be a decrease in signal, but overall, the assay results should not be affected. Storing the samples for extended periods of time is not recommended, as it could lead to further reductions in signal.

Do NOT invert the plate to dump the liquid. The beads will not adhere to the plate. After centrifugation using a swing bucket rotor with a plate adaptor, you can remove the liquid by using a pipette.

Typically flow cytometers generate output files in FCS format (e.g. FCS 2.0, 3.0, or 3.1) and in some cases in list mode file format (LMD). FCS files can be analyzed by different software. Data generated from using LEGENDplex™ kits can be analyzed using the LEGENDplex™ data analysis software freely available on the BioLegend website (biolegend.com/legendplex/software). A free of charge software license dongle is currently provided in the kit. Please check our website for the most updated versions of the software.

Filter plates or V-bottom plates have been included in some kits for your convenience. Performing the assay with filter plates is generally recommended. A vacuum filtration unit is required to work with the filter plates. If however, you don’t have access to a vacuum manifold, then you can use the V-bottom plates or micro-FACS tubes as described in the manual and follow the recommended assay protocols for the type of plates or tubes you choose. All tubes and plates should be made from low binding polypropylene. Polystyrene ELISA or cell culture plates should not be used.

Yes, all of the above sample types may be used with LEGENDplex™ kits as long as they are prepared in a manner compatible with immunoassays. For tissue homogenate samples, they should be prepared in a neutral pH buffer containing no denaturing chemicals (e.g. SDS, urea, thiourea, cholate, etc.) and no ionic detergents and minimal amounts of non-ionic detergents (e.g. NP-40) may be used in sample preparation. The tissue/cell homogenization buffer should not contain excessive ionic strength above physiological concentrations of salts. If possible, the buffer should also contain protease inhibitors. In addition, the samples should be centrifuged to remove particulates.

LEGENDplex™ assays can be used on most commonly used flow cytometers that can read APC and PE, such as BD FACSCalibur™, BD FACSCanto™, BD FACSCanto™ II, BD LSR I™, BD LSR II™, BD LSRFortessa™, BD FACSAria™ I, II, III, BD Accuri C6™, BD FACSVerse™, Gallios™, CytoFLEX™, Moflo XDP™, Attune™NxT, NovoCyte™, MACSQuant®, and Guava® easyCyte. Please refer to the “Materials to be provided by end-user” section of your LEGENDplex™ manual for details on the requirements of the machines in terms of laser and channel configurations. For other brands of flow cytometers the end-user needs to make sure that the machine is set up properly before use.

The LEGENDplex™ data analysis software uses a default fiveparameter logistic curve-fitting algorithm, which determines sample concentrations and calculates minimum and maximum detection concentrations of the standard curve. If the default algorithm does not work for a particular set of data, other curve fitting methods are available in the Options of the software. If sample concentrations fall outside of the maximum detectable range, the sample will have to be diluted and re-analyzed.

LEGENDplex™ and Luminex® are both bead-based multiplex immunoassays that use the basic principles of sandwich immunoassays. Both systems use fluorescence-coded beads to achieve multiplexing. The major difference is in the data acquisition. LEGENDplex™ uses common lab flow cytometers for data acquisition, whereas Luminex®-based assays have dedicated machines for this purpose. Therefore, one of the advantages is that LEGENDplex™ can be run on widely available flow cytometers without needing specialized machines. As such, LEGENDplex™ cannot be run on Luminex® machines.

If a single laser instrument is used for both reporter (FL2 or PE) and classification (FL3), then compensation is needed to resolve the signal spillover from FL2 to FL3, and vice-versa. Even on dual laser instruments, some compensation may be required. Please check your machine for the need for compensation and when required use the calibration beads provided and follow the Flow Cytometer Setup instructions in the LEGENDplex™ manual. In addition, adjust the PMT voltages such that there is good bead separation, the bead populations are not out of scale, and the PE signal is appropriately amplified to ensure proper signal sensitivity.

LEGENDplex™ kits are valid for 6 months from the date of receipt. 

Yes. It is possible to use the non-linear part of the standard curve for calculation of results. The LEGENDplex™ data analysis software uses a five-parameter curve-fitting algorithm, which determines the minimum and maximum detection concentrations of each target and reports them. If sample concentrations fall outside of the maximum detectable range, the sample will have to be diluted and reanalyzed.

Yes. We now have software for both Macs and PC. Get more details and download the software at: www.biolegend.com/legendplex/software.

We have found that fixing/sterilizing in samples 1-4% paraformaldehyde in 1X PBS prior to reading the samples out on a flow cytometer is compatible with our LEGENDplex™ reagents. However, incubation time with PFA should not exceed 10-15 minutes. Immediately following the fixation, the PFA should be washed out using the 1X Wash Buffer supplied with the kit. Beads should be resuspended in the 1X Wash Buffer supplied with the kit. Prolonged incubation of beads with 1-4% paraformaldehyde will decrease the signal dramatically.

Yes, most fixation reagents are fine to be used with Zombie dyes. However, it should be noted that Zombie dyes can still be sensitive to reactive oxygen species. Light exposure or reagents with hydrogen peroxide can lead to free radical formation, affecting fluorescence.

While we typically do not test Zombie Aqua™ with the UV laser, its excitation peak suggests it is effectively excited at 355 nm. However, we would not recommend using BV510™ off the violet laser and Zombie Aqua™ off the UV laser at the same time. Due to cross-beam excitation of BV510™ by the UV laser and the violet excitation of Zombie Aqua™, this would lead to significantly increased background and excessive compensation requirements.

Zombie dyes that have been tested for microscopy applications in-house will display data on the product webpage. It should be noted that Zombie may not work for dead cell discrimination in every microscopy application, as a complicated point will be to determine the level of Zombie signal that constitutes a dead cell. Another difficulty may be finding the proper plane for microscopy in order to observe the dead cells.

Yes, Zombie can be used with Annexin V to discriminate live, apoptotic, and dead cells. Cells double positive for both Zombie and Annexin V are dead, while Zombie-dim/Annexin V-positive cells are apoptotic. Live cells will be Zombie-low and Annexin V-negative. The advantage to Zombie over PI and 7-AAD is that you can now fix and/or permeabilize the cells to stain for cell surface and intracellular antigens.

Zombie dyes have been tested against other leading competitors' fixable viability kits and given comparable results. We also highly recommend that you titrate down the amount of each dye used in order to best match the negative signals of your unstained sample and MFI- (mean fluorescence intensity) stained samples.

It is made in E. coli, covering human aa Met1-Asp320.

The fixation process can contort and alter the membrane of cells, effectively rendering them as dead. Since the ability of Zombie to stain dead cells is correlated with cell permeability, your results may no longer be a valid representation of dead versus live cells.

These dyes bind in equilibrium with DNA. Therefore, external dye concentration must be maintained during analysis and the dye should not be washed out.

We have tested mouse CD4 positive cells that have been isolated from the spleen and lymph nodes with directly conjugated Nanobeads, as well as CX3CR1 expressing cells from mouse bone marrow. The cells appear unaffected as assessed by migration, differentiation and stimulation assays.

There are no restrictions for negatively selected cells, as they are untouched by antibodies. For positive selection, there may be clones that are less efficient than others due to possible epitope competition. Please refer to the Technical Data Sheet or contact the Technical Service Group for advice.

We have not tested this application yet.

Yes, this is possible, but if you are staining for the same marker as used for sorting, we would recommend using a different clone with non-overlapping epitopes.

Yes, they can be sterile filtered as the particles are smaller than 0.22 µm.

Not recommended, however lyophilized particles can be made available as a custom product.

No, not currently.

Yes, we do. Please contact our Custom Solutions Team at cst@biolegend.com.

We have not determined this yet.

No, not currently.  We have found that cells are functional without the need to detach the magnetic Nanobeads.

Hydrophilic polymers.

The diluted, 1X MojoSort™ buffer contains 1X phosphate buffer saline (PBS) supplemented with 2 mM EDTA and 0.5% BSA. The 5X solution is sterile. To preserve sterility dilute with sterile water under sterile conditions.

1 to 2 years depending on product type.

The average diameter is approximately 130 nm.

The particles are stored in a neutral pH solution containing BSA and sodium azide.

It is possible that other magnetic separation systems can be used. To learn more about this please contact our Technical Service Group.

1. the early termination of the translation of epitope-tagged protein;
2. non-specific detection with anti-epitope tag antibody due to no/poor blocking of the immunoblot.
3. partial degradation of the protein
4. the concentration of the primary or the secondary antibody is too high.
5. Washing between the incubations is not efficient

It is likely the target protein was not tagged or the tag is out of the reading frame or the protein is degraded.

The commonly used applications are western blotting, immunoprecipitation and protein purification. These can also be used for immunofluorescence microscopy and flow cytometry.

• Tag can be easily and rapidly added to a known gene.
• Multiple tags can be added if required.
• Well-characterized antibodies are available.
• The antibody is specific to the tag, therefore cross-reaction with other proteins is avoided
• Proteins and protein complexes can be purified using standardized practices.
• Tagged proteins can be distinguished from otherwise identical untagged proteins
• Possible to study novel and poorly immunogenic proteins.

A few can be as follows

• A cloned and characterized gene or cDNA must be available
• The epitope tag may interfere with protein structure or function
• Epitope-tagged genes can be expressed at abnormal levels due to the use of heterologous promoters
• The epitope-tagged gene must be introduced into the cell, tissue, or organism of interest.

A lack of signal following western blotting may indicate a few different problems.

1. No or very poor transfer. This can be addressed by quickly checking the membrane with Ponceau-S staining.
2. The expression level of the epitope-tagged protein may be too low. Load more ug quantity of the sample and include a positive control.
3. A very diluted antibody may be the problem, try a few different concentrations of the antibody to probe the western blot.
4. Also, a remote possibility is that the epitope tag is out of frame and is not expressed resulting in a lack of any signal.

Once the products are available on BioLegend's website, contact BioLegend's technical service team for any product questions.

Antibodies used are different in different kits. The specificity of the antibodies partially dictate how much signal is being detected.
Recombinant standards used are different.  First of all, different kits may use recombinant proteins expressed and purified using different method. Second, recombinant proteins expressed from E. coli from the same source can show greater than 10 fold difference in term of immunoreactivity from lot to lot, primarily due to refolding inconsistency. Third, different kit standards can be produced and calibrated against different references. So far there is no universally accepted standardization for cytokine immunoreactivities.
Each BioLegend ELISA product was developed and validated with reagent concentrations and protocols optimized for best analytical robustness.  Any changes to the reagents (standards, antibodies, matching matrices) and protocols etc all affect the final assay performance.

Specific activity will vary for each lot and for the type of experiment that is done to validate it, but all passed lots will have activity within the established ED50 range for the product and we guarantee that our products will have lot-to-lot consistency. Please conduct an experiment-specific validation to find the optimal ED50 for your system.

Our testing shows that the recombinant proteins are able to withstand room temperature for a week without losing activity. In addition the recombinant proteins were also found to withstand four cycles of freeze and thaw without losing activity.

Our carrier-free recombinant proteins are shipped on blue ice.  These products have been validated to maintain activity after shipping using blue ice.

We quality control each and every lot of recombinant protein. Not only do we check its bioactivity, but we also compare it against other commercially available recombinant proteins. We make sure each recombinant protein’s activity is at least as good as or better than the competition’s. In order to provide you with the best possible product, we ensure that our testing process is rigorous and thorough. If you’re curious and eager to make the switch to BioLegend recombinants, contact your sales representative today!

BSA is used as carrier proteins to improve the stability of the reconstituted protein, and to avoid the product from sticking to the wall of the vial.

Our animal-free products are proteins that go through the entire production process without touching any animal containing components. This includes using animal-free media and purification equipment that are animal component-free. Some studies which are particularly sensitive to contamination by mammalian pathogens may require the use of animal-free products. Our carrier-free products do not contain any carrier protein in the solution, as expected, but they are produced using animal-containing components. Both versions are expected to have similar activity and function, though specific activity is lot-dependent.

There is no direct relationship between International Units and the units that are calculated using the inverse of the specific activity because those units are not calculated using the International Standard provided by WHO (National Institute for Biological Standards and control). In those cases, the best way to compare the activity of two sources of recombinants is by doing the bioassay side by side using the same system.  If our protein has been tested using the International Standard, the International Units will be indicated on the technical data sheet for that product.

All our carrier-free and animal-free formats of recombinant proteins do not have any additional carrier proteins such as BSA in the formulation. Typically our ELISA standard recombinants have carrier proteins added to the formulation for added stability and to avoid the product from sticking to the wall of the vial. When the presence of carrier is not desirable (e.g., in-vivo applications), carrier-free proteins can be used directly. When carrier proteins do not affect the outcome in a study, the customer can decide what type of carrier protein they would like to use and whether it is necessary to add it to their stock.

The specific activity range of the protein is indicated on the product datasheets. Because the exact activity values on a per unit basis can largely fluctuate depending on a number of factors, including the nature of the assay, cell density, age of cells/passage number, culture media used, and end user technique, the specific activity is best defined as a range and we guarantee the specific activity of all our lots will be within the range indicated on the datasheet. Please note this only applies to recombinants labeled for use in bioassays. ELISA standard recombinant proteins are not recommended for bioassay usage as they are not tested for these applications.

Most of our carrier-free recombinant proteins are shipped in liquid form, so there is no need for reconstitution. If you need to make dilutions, refer to the formulation on the product datasheet. Stock solutions should be prepared at no less than 10 μg/mL in buffer containing carrier protein such as 1% BSA or HSA or 10% FBS (for chemokines, use either BSA or HSA). For long-term storage, aliquot into polypropylene vials and store in a manual defrost freezer. Avoid repeated freeze/thaw cycles.

For reconstitution of our lyophilized recombinant proteins (ELISA Std, animal-free, and some carrier-free) please refer to the Certificate of Analysis and/or Technical Datasheet included with the product and follow the instructions. 

0.5 μg/ml to 4 μg/ml range can be used for titrations.

* Transfer buffers may have become contaminated.
* Post-antibody washes may not have been performed with sufficient time or volume.
* Blocking and incubation agents were not freshly prepared or were too dilute.

*  Transfer efficiency may have been poor. Check protein transfer by staining the gel and/or membrane.
* Incorrect storage of antibodies or ECL western blotting detection reagents.
* Insufficient protein may have been loaded on the gel. Depending on the location of the target protein, membrane or nuclear preparations may be required (instead of whole cell lysates).
* Film exposure time may have been too short.

We don't test this in house. Typically the molecular weight of an IgG antibody is approximately 150kd.

The size of the target protein can be affected by many factors such as:

• Post-translational modification such as phosphorylation, glycosylation can increase the size of the protein.
• Post-translation cleavage - e.g. some proteins may get cleaved to the active form from pro-protein form via post translational cleavage.
• Different sized proteins produced from the same gene can be produced by alternative splicing (Splice variants).
• Overall charge on the protein based on amino acid composition can also affect the protein migration in a gel.
• Strong protein-protein interactions can potentially lead to Multimers thereby leading to the appearance of higher size bands. This is usually prevented in reducing conditions and boiling of the sample before loading to the gel.

Due to their vulnerability to photobleaching, these fluors are not recommended for IF. Dyes such as Alexa Fluor®, DyLight™, and BV421™ are good alternatives for the IF application.

Typical concentrations of monoclonal antibodies for use in IHC are from 5-25 µg/ml. Polyclonal antibodies can be used at a range of 1-10 µg/ml. We will indicate on the datasheet if an antibody has been tested in-house or published for use in this application. In addition, you can do a lit search with the clone name and immunohistochemistry/paraffin/frozen to see what the protocol details are.

Anti-fade should not be added for live cell imaging as it will deprive the cells of oxygen.

No, due to spectral emission overlap you will not be able to distinguish between GFP and AF488/FITC

In most cases, this is true. Antigen retrieval helps both the accessibility of the antibody to the tissue and also counteracts the fixation effects on the recognized epitopes. Check the application references for any additional details for IHC or IF experiments.

If an antibody is listed as being quality-tested or validated for IHC, we can look up the protocols for you. If this application is reported in literature for IHC, we have not specifically tested the product for this application. It relies on information from publications. Of course, we seek to provide the most updated information on these publications, so we can update our datasheets if the information is incorrect.

The sequence of the UV-labile peptide is not needed to use the reagent. MHC molecules are not stable without a peptide, so these peptides are used just for two purposes: stabilize the MHC molecules and serve as a place holder to be substituted by the peptide of interest.

Yes, please contact our Custom Solution Team with your request at cst@biolegend.com, or contact your local BioLegend representative.

Not currently.

Follow the protocol for HLA class I ELISA. An assay positive control is provided (Cat#280301) that can be diluted to a high, medium, and low concentration. Signal intensity can be correlated to affinity of the peptide.

There are a number of databases and webpages that can help, these are three of them:

http://www.iedb.org/
http://tools-int-01.liai.org/main/
http://www-bimas.cit.nih.gov/molbio/hla_bind/

Please see the linked document titled “Peptide Sequences”. The document contains a table with commonly used peptides by allele to study antigen specific T cells.

With the traditional pre-assembled Tetramer approach, this is difficult to do, and not cost-effective. With Flex-T™ technology, as there is more flexibility to assemble the tetramers, it is easy and affordable to screen a sample for several specificities². To facilitate this approach, a combinatorial color coding system has been developed. Please visit the Flex-T™ webpage for a detailed description.

2) Hadrup SR et al. Parallel detection of antigen-specific T-cell responses by multidimensional encoding of MHC multimers. Nat Methods. 2009 Jul;6(7):520-6.

Yes, the same monomer can be assembled with different Streptavidin conjugates, providing great flexibility for color choices. Additionally, it can be stored frozen and the tetramer assembled shortly before the experiment. This increases the storage time of the reagent.

There are no special requirements for the peptides. Peptides that naturally bind to MHC molecules will bind to Flex-T™ reagents. For class I molecules, typical length is about 8 – 10 amino acids. Class II molecules accommodate longer peptides, about 14 – 20 amino acids¹.

1) Mohan JF and Unanue ER. Unconventional recognition of peptides by T cells and the implications for autoimmunity. Nat Rev Immunol. 2012 Oct;12(10):721-8.

The T-cell mediated innate immune response is defined by the interaction between antigen presenting cells and T cells, through the Major Histocompatibility Complex (MHC) and the T cell receptor (TCR). MHC molecules present a peptide to antigen-specific T cells that recognize this peptide. Soluble, monomeric MHC molecules bind very weakly to the TCR. However, by making a tetramer through a fluorescently labeled streptavidin conjugate, the complex binds to several TCRs, creating a more stable interaction and making it useful for flow cytometric detection of antigen specific T cells.

Long-wave UV, 366 nm, 8 Watts (We recommend, for example, CAMAG cat# 022.9115, or Ultraviolet Crosslinker CL-1000). The distance from the solution to the light source should be 2 – 5 cm (approximately 0.8 – 3 inches).

Flex-T™ (Flexible-Tetramers) is BioLegend’s brand name for our Soluble MHC product line. It encompasses monomers, the ultraviolet (UV) peptide exchange technology, and all associated products and applications.

Flex-T™ MHC monomers are loaded with a peptide that can be degraded by the use of a UV light source. This allows for a peptide exchange when the UV irradiation is done in the presence of the peptide of interest (which is not UV-labile). This flexibility permits the screening of virtually any peptide of interest with enough affinity for the MHC allele that it is loaded onto.

They are treated with our proprietary mixture prior to lyophilization.

Yes, it should be possible, although this has not been verified by BioLegend.

For this, use Veri-Cells™ Activated (Cytokine) PBMC which have been activated with Cell Activation Cocktail (with Brefeldin A) containing PMA and Ionomycin. These have been validated to stain positive for activation-induced cytokines including TNF-α and IFN-γ. Veri-Cells™ PBMC and Veri-Cells™ CD4-Low PBMC will not have detectable TNF-α or IFN-γ.

Each Veri-Cells™ product is tested with a panel of antibodies to ensure expression of these markers falls within the expected range.  For more information regarding the exact markers used for each product, please contact technical service.

Depending on application pursued it may require diluting the antibodies prior to use.

Yes, the reagent is designed for drug screening work and other situations that require DMSO.

You can dialyze or desalt the enzyme into a phosphate-free buffer.

This is almost certainly caused by inadequate mixing of the Stabilizer. This results in a high background signal because of non-enzymatic decay of ATP substrate. The Stabilizer is added in a relatively small volume (20μL), and the operation of pipetting up and down with a pipette set to 20μL volume may not result in sufficient mixing when the total volume is 270μL. Try pipetting up and down while stirring at the same time. Alternatively, add the Stabilizer with one pipette set at 20μL volume and mix using a larger pipette set to ~150μL volume. This ensures thorough mixing of the Stabilizer solution with minimal effort.

Purify the DNA from your sheared chromatin samples and run the DNA on a 1-2% agarose gel. DNA should be sheared to a range of 100-900 bp. Below is an example of chromatin from a number of different cells lines that has been sheared optimally using enzymatic digestion compared to chromatin which was over-digested.

Lane 1: Ladder, Lane 2: HeLa, Lane 3: Jurkat, Lane 4: NCCIT, Lane 5: HeLa IL-6	Lane 1: HT1080+ IFNγ, Lane 2: NIH353, Lane 3: HT29, Lane 4: HCT116, Lane 5: Ladder

Sheared chromatin may be saved at -80°C prior to performing the immunoprecipitation. If you are saving the chromatin, we would recommend aliquoting the chromatin as it can be sensitive to freeze thaw cycles. Alternatively, purified DNA can be saved prior to downstream analysis.

Sonication may be ideal for difficult to lyse cell types and provides random fragmentation but may damage epitopes. It is not suitable for Native ChIP experiments (in which the chromatin has not been cross-linked). Enzymatic digestion is milder and can be used in Native ChIP experiments but will exhibit sequence bias and may not be suitable for hard to lyse cells.

Human and mouse cell lines such as HeLa, Jurkat, Daudi, THP-1, NIH3T3, and 293T have been validated using this protocol. Other cell types (plant, yeast, tissue, FFPE samples, etc.) may require optimization.

An important consideration when performing ChIP is the amount of chromatin that will need to be loaded to the column in order to elute sufficient IP’ed DNA for downstream analysis. Sufficient cell numbers should be used so that at least 3µg of chromatin can be used per IP. Start with 1-15 million cells. However, the cell number can be scaled up and the reagents need to be adjusted accordingly.

This should be determined empirically by the end-user. Refer to the datasheet for our Go-ChIP-Grade™ Purified Antibodies, or other ChIP-validated antibodies. The suggested dilution of the Go-ChIP-Grade™ anti-RNA Polymerase II Antibody; Clone 8WG16 (positive control) is 1:300 – 1:500 by volume.

The quality of the ChIP antibody has a major impact on the success of the assay. Use only ChIP-validated antibodies. Make sure to have good quality chromatin samples with fragments between 150-900bp. Insufficient DNA shearing leads to longer DNA fragment length that can cause high background in downstream experiments. Insufficient wash steps can also leave traces of non-specific chromatin alongside enriched DNA. If background remains high, include an additional wash step during the IP protocol.

Make sure to only use ChIP-validated antibodies and follow the instructions for the amount of antibody to be used or titrate the antibody for your experiment. It is also recommended to include ChIP validated positive and negative controls (included in the kit) to validate the efficiency of your ChIP assay. Good quality chromatin samples and chromatin fragment sizes between 150 – 900 bp are also critical. Other factors can include whether cells were effectively lysed, over-fixation and reduced antibody binding due to decreased epitope availability, and insufficient starting material (chromatin sample per IP).

TotalSeq™ is BioLegend’s brand of antibody-oligonucleotide conjugates, to enable simultaneous analysis of proteins and mRNA in single cells. CITE-seq and REAP-seq are two similar workflows to do simultaneous protein and mRNA analysis, and the TotalSeq™ conjugates integrate seamlessly into these workflows.

Hashing allows customers to run multiple samples in a single lane of a 10x Genomics Chromium instrument, or equivalent, which optimizes the number of cells or samples that can be analysed simultaneously. This can reduce variability due to batch effects, handling, etc. It can also improve yield when cell numbers are low and optimize the use of the platform.

We have not tested this, but there is no reason to believe that non-competing clones for the same target would be problematic. Similarly, a sub-saturating concentration of both reagents against the same target should be tolerated by the cell/technique. The original CITE-seq paper by Stoeckius et al.(Fig. 2) demonstrated that cells can be co-stained for downstream analyses. Although the authors did not use current TotalSeq™ reagents, the technology is the same. In addition, other CITE-Seq users have also demonstrated this approach. Please contact our technical service group for more information.

The oligo should withstand sorting. However, based on theoretical considerations we recommend to use non-competing clones, to minimize impact in either FACS or sequencing signal/reading. Even if exposed for a very short time, we have not evaluated the effect of ultraviolet or violet lasers on the oligo conjugates. An alternative to flow sorting could be magnetic bead enrichment, such as our MojoSort™ platform.

A published article multiplexed 82 antibodies conjugated to oligonucleotides. The authors did not use current TotalSeq™ antibodies, but the method is very similar, which they termed REAP-seq. There is no reason to believe that larger panels are not possible.

This depends on the biological question that needs to be answered. From the technical perspective, when using 10X Genomics Chromium, it is recommended to load 5,000 – 10,000 cells per lane. The use of Hashtags can increase that number. For ease of staining, we typically recommend 1 million cells, but this number also depends on availability of cells and the operator’s ability to handle low cell numbers.

This has to do mostly with antibody affinity, titration, and level of antigen expression. BioLegend and some collaborators are working on titration data. There is also some data already published. In theory, as long as the antibody can bind one molecule, PCR amplification and sequencing should pick it up, but there is always background noise. A common way to control for this is to add negative cells to your experiment. For example, if working with human PBMCs, use mouse cells and vice versa.

No, sequencing as the final readout is not affected by cell autofluorescence.

This is a technically challenging application as micro-RNAs are very small, and in many cases will be as small as or even smaller than the required primers to execute the protocol. It is not possible at the moment.

Not in the same experiment; it is not possible because CyTOF® requires its own instrument and protocol, and it destroys the sample in the process. To do so, it is necessary to split the sample and run both CyTOF® and CITE-seq assays. Note that CITE-seq generates equivalent data for surface proteins as CyTOF® with higher panel multiplexing capabilities and at a single-cell level.

The oligo is attached to the antibody in the same method as some of our fluorophore-conjugated antibodies that are quality tested for flow cytometry. Once the antibodies have been conjugated to the oligonucleotide, we verify by flow cytometry that the antibody can still bind to its target. This is part of our quality control process for TotalSeq™ conjugates.

If the populations of interest can be clustered/identified without sorting, the likelihood that you need to pre-sort is minimal. However, if the cell number in the cluster is very small, it may not be sufficient to draw conclusions when compared to control, or other cell populations. In which case, enrichment of this population prior to CITE-seq analysis may help with obtaining more meaningful sequencing data that is selective for your rare cell population(s) of interest. We also recommend to sort out dead cells if the viability in a sample is lower than 95%.

Running dead cells during CITE-seq essentially leads to “wasting” single cell runs on dead cells, which may ultimately lead to sub-optimal data, or not processing enough cells to pick up the positive events, especially if the frequency of your target cell is very low. Dead cells can also be removed using magnetic particles. If performing CITE-seq before eliminating dead cells is required, it is possible to use bioinformatics methods to try to clean up low quality events (which may include dead cells). This could be a more complex approach, but in such cases, we recommend the following reading:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4758103/
https://www.ncbi.nlm.nih.gov/pubmed/28045081

We are still in the process of identifying optimal concentrations for the use of TotalSeq™ reagents. However, here is what we can recommend for titrations:

1. Performing titrations using the actual CITE-seq method is costly, but using Hashtags can make the process more affordable. See Fig. 3 and associated methods of this publication for more information.
2. Perform flow cytometry experiments to find the optimal antibody amount, using the same clone conjugated to PE. The flow cytometry antibody amount translates well to the CITE-seq amount.
3. Label the cells with different concentrations of TotalSeq™ antibody, and then use a poly-dT oligo conjugated to a fluorophore, such as Alexa Fluor® 647, as a secondary reagent to detect the TotalSeq™ antibody. This is a more direct method as compared to the ones listed above, but it requires the use of the actual TotalSeq™ antibody.

To provide flexibility when sequencing. Libraries can be mixed at different ratios before sequencing depending on the number of reads needed.

There are several companies offering primer synthesis. We can recommend IDT technologies. For additive primers, desalt purification is sufficient. For index primers, either HPLC or PAGE is needed.

Cell Ranger 3.0 can be used for barcode count, although some reagents or applications may not be supported by 10x Genomics. Useful resources include CITE-Seq-Count, and the Satija Lab. Please contact our technical service group (tech@biolegend.com) for further information.

Hashtag reagents are intended to be used for sample barcoding, which allows users to combine multiple samples into a single lane, then de-multiplex during analysis. Instead of select antigen-specific antibodies, the hashtags are designed so that they are specific for human or mouse cells, and to cover as many cells as possible. For human samples, the hashtags are made of two antibodies that recognize ubiquitous surface markers, CD298 and β2 microglobulin, each conjugated to the same oligonucleotide containing the barcode sequence. For mouse samples, the surface markers are CD45 and H-2 MHC class I. The conjugates are already pre-mixed and ready to use.

Keeping stained cells in any fixative for longer periods of time is not recommended. You can use Fluorofix™ buffer as recommended on the data sheet, followed by washing with cell staining buffer and then at the last step, resuspend your fixed cells in cell staining buffer till you acquire your samples in a day or two.

It is not recommended. It is best to use PBMCs for this testing.

Generally, Brefeldin A is more toxic for longer term incubations, so for shorter stimulations (6 hrs or less) use Brefeldin A.  For longer stimulations use monensin.  We recommend optimization for each cell type and protocol.

We would not recommend extended storage of the product after dilution.  It is best to prepare the dilution as needed on the day of use.  Because there are no preservatives in these buffers and they could be susceptible to changes in pH, long term storage greater than a day or two is not recommended.

We recommend to thaw in warm water after removing from the fridge.  The Brefeldin A is dissolved in DMSO, which freezes at 18°C, so this is normal.  No one has reported any problems with the freeze/thaw cycles and this is how we recommend it is stored.   If customers are concerned, they can aliquot the Brefeldin A to avoid the freeze thaw.

Brefeldin A is provided at a 5mg/ml concentration.

Monensin is provided at a 2mM solution.

FluoroFix™ (cat# 422101) is designed for fixation of cells stained with fluorescent labeled antibodies, including tandem dyes.

Annexin V binding requires the presence of calcium in the solution.  So, we provide Annexin V Binding Buffer (cat # 422201), which is optimized for the best performance of Annexin V staining.

The plates are not sterile. You would handle them as you would handle most typical flow cytometry staining protocols.

Yes. Customers can use half of the plate or whatever specificities they are interested in. However, the whole plate should be reconstituted. The half plate of antibodies must be transferred to another empty plate for the staining. The remaining half must be sealed and stored at 2 - 8°C in the dark and used within a month.

Yes.

For more info, visit: biolegend.com/custom_solutions.

The kit should be stored at 2 - 8°C upon receipt. Once opened, the plates must be reconstituted immediately. Reconstituted plates can be used or stored at 2 - 8°C sealed in the dark and used within a month.

The antibody is in a one test per well format. There will not be any antibody left if the full test is used. Customers may decide to use less than the recommended volume per test, but this is not recommended and the performance is not guaranteed. Customers may also selectively transfer certain antibodies from the original plate to a new plate and use after reconstitution. If any antibody is not used after reconstitution, the plate can be sealed and store at 2 - 8°C for a month in the dark. Once reconstituted, re-drying is not recommended as this may result in a loss of signal.

Yes, as long as the fluorophores on these antibodies are compatible and proper compensation has been applied during acquisition and analysis.

This may work with lower numbers of total cells, but we recommend trying to keep higher concentrations of cells for faster analysis. Of course, how many cells are needed depends on the specific application. Successful staining has been done with 1 x 10⁵ cells/well.

Lyophilized product has a guaranteed shelf life of 6 months unopened. Reconstituted plates can be used or stored at 2 - 8°C sealed in the dark and used within a month. Be sure to properly seal the plates to prevent evaporation and shield the antibodies from light.

If the protocol is followed closely, the variability should be minimal. The variability should be similar to single vial antibody staining.

Currently, we do not offer any automated analysis solutions for LEGENDScreen™ kits. However, a commercial solution is available from Astrolabe Diagnostics. The Antibody Staining Data Set is an example application of the LEGENDScreen™ Human PE Kit (see Amir et al.).