BioLegend's LEGENDScreen™ products are lyophilized, fluorophore-conjugated antibodies provided in 96-well plates for the purpose of screening cell surface molecules on your cells of interest.
Why choose LEGENDScreen™?
Large selection of specificities per screen - Human PE Kit has 361 cell surface marker antibodies plus 10 isotype controls. Mouse PE Kit has 255 cell surface marker antibodies plus 11 isotype controls.
Fast and easy to follow protocol.
Directly conjugated antibodies at pre-titrated optimal concentrations provide reliable results.
Full kit with staining buffer, fixation buffer, and plate sealers.
Introducing LEGENDScreen™ PE Kit (cat # 700005). This kit contains 255 PE-conjugated monoclonal antibodies against mouse cell surface markers, plus 11 mouse, rat, and hamster Ig isotype controls. All of the antibodies have been pre-titrated to optimum concentrations, arrayed on three 96-well plates, and lyophilized in a ready to use format. LEGENDScreen™ is the most cost-effective way to test this many directly-conjugated antibodies in a single assay. This kit can be used to screen cell lines and primary cells (splenocytes, bone marrow-derived cells, and cells isolated from tissues, etc.). The workflow is simple and easy. Just reconstitute the antibodies, add your cells and then analyze. Cell staining is done directly in the plates, so no additional transfer of reconstituted antibody is required.
Blank wells on the plates allow for adding your own markers. This format allows users to screen cells of interest directly or co-stain with other markers labeled with different fluorochromes. Positive hits from the screening can be quickly identified based on the plate maps in the manual or online, with detailed clone information. Individual fluorochrome conjugated antibodies should be used to confirm the screening results.
The LEGENDScreen™ Mouse PE Kit provides a convenient, easy to use, and powerful tool for immunology, stem cell, and cancer research.
FAQs
What is the level of variability from one experiment to the other? If the protocol is followed closely, the variability should be minimal. The variability should be similar to single vial antibody staining.
How should the kit be stored? 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.
How do I request a custom LEGENDScreen™ product with only my specificities of interest? For more info, visit: biolegend.com/custom_solutions.
What are the guarantees regarding the lyophilized plate compared to the reconstituted plate? 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.
I have added my own antibody solution to the lyophilized product, will the lyophilized antibody work? Yes, as long as the fluorophores on these antibodies are compatible and proper compensation has been applied during acquisition and analysis.
I am not going to use all the reconstituted antibody solution. Can I keep the leftover for later or re-dry the solution in the dark? 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.
If I don't have enough cells and use less than 4 x 106/ml (3 x 105 cells/well), will it still work? 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.
Are these plates made under sterile conditions? The plates are not sterile. You would handle them as you would handle most typical flow cytometry staining protocols.
Can I use half or less of the plate and keep the rest for later? 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.
For more answers,
Plate Preparation
One hour before the staining, perform the following steps to prepare the plates:
Remove the lyophilized plates from the aluminum pouches.
Centrifuge the plates at 600 × g for 10 minutes.
Make sure the lyophilized cakes settle to the bottom of the plates. Keep the plates upright at all times from this point forward. Handle plates with care so that the cakes are not agitated at any time.
Gently settle the plates on the bench away from drafts and air conditioning vents. If possible, discharge any static electricity on yourself prior to removing the cover. Hold the plate firmly and carefully remove the plate cover from one corner, one well at a time, for easy opening and to prevent cross-well contamination. You may need to wiggle the cover a little while pulling back slowly.
Discard the cover. Do not move the plate, reseal with the original cover or apply any sealer until the lyophilized antibodies have been reconstituted.
Reconstitute the lyophilized antibodies immediately, with 25 µL/well of deionized water using a multichannel pipette. To avoid cross-contamination, do not let the pipette tips touch the wells. Make sure that all the cakes are dissolved in water. If a cake is stuck on the side of the well, aspirate the 25 µL water added to the well and rinse the cake down to the bottom.
Seal the plates with the plate sealers provided.
Wait at least 15 minutes before the staining procedure. Keep the plates in the dark.
Notes:
PE-conjugated antibodies are light-sensitive. Try to minimize the exposure of the plates to light as much as practically possible.
Do not open the pouches until the day you are ready to run the experiment. Once the plates are removed from the pouches, the antibodies must be reconstituted immediately.
If an experiment is not performed after reconstitution, plates can be sealed and stored in the dark at 2 - 8°C for up to one month.
Preparation of Cells
Obtain desired tissue (e.g. leukocytes, cell lines or tissue isolates) and prepare a single cell suspension. Wash cells in 1X PBS or cell culture medium of choice, and resuspend in Cell Staining Buffer at a density between 4 × 106 and 1 × 107 cells/mL.
For cultured cells in suspension, spin and resuspend cells in Cell Staining Buffer at a density between 4 × 106 and 1 × 107 cells/mL.
For cultured adherent cells, dissociate cells using a mild enzyme (e.g. Accutase® Cell Detachment Solution, Cat. No. 423201) or non-enzymatic dissociation buffer. Wash cells in 1X PBS or cell culture medium of choice, and resuspend in Cell Staining Buffer at a density between 4 × 106 and 1 × 107 cells/mL.
Filter the cells through a 40 µm cell strainer to remove any clumps. Keep the cells on ice before use. Approximately 22.5 mL of cells at a density between 4 × 106 and 1 × 107 cells/mL are needed for all three plates. Lower cell density (e.g. 1.5 x 106 cells/mL) might be used depending on the application.
Staining Procedure
Using a multichannel pipette, add 75 µL of cells (~ 3 - 7.5 x 105 cells/well) to each well of the plates.
Set up extra tubes to stain cells for flow cytometer setup and compensation, if needed.
Using the multichannel pipette, gently mix the cell suspensions by pipetting up and down 2 - 3 times. Be sure to change tips between each row or column. Avoid creating bubbles while pipetting.
Incubate for 20 - 30 minutes at 2 - 8°C in the dark.
Spin the plates at 500 × g for 10 minutes to pellet cells in each well. Immediately after centrifugation, dump the supernatant into the sink by quickly inverting and flicking the plate. Gently blot the plate on a clean paper towel, being careful not to disturb the cell pellet.
Using a multi-channel pipette add 200 µL of Cell Staining Buffer to each well. Gently mix up and down to resuspend cells. Be sure to change tips between each row or column.
Repeat step 5.
To fix the cells, using a multichannel pipette, aliquot 100 µL of Fixation Buffer into each well. Gently mix up and down to resuspend cells. Be sure to change tips between each row or column.
Incubate for 10 minutes at room temperature in the dark.
Repeat steps 5 - 6.
Repeat step 5 one more time.
Resuspend cells completely in 160 µL of Cell Staining Buffer per well and analyze using a flow cytometer. We recommend acquiring 70 µL of sample and collecting 5,000 - 10,000 events. Users should determine the optimal number of events to be collected based on specific application they are testing. While the first plate is being acquired, store the other plates at 2 - 8°C in the dark.
Tips for Successful Staining
Read the entire manual carefully before the experiment.
Plan the experiment in advance. Designate a full day for this experiment. Do not rush any step.
Make sure that the flow cytometer’s autosampler is well maintained and working well before the experiment. If the flow cytometer does not have an autosampler, the samples from each well of the plates should be transferred to individual FACS tubes and tube adjust the sample volume to approximately 300µl or more to avoid running dry. Alternatively, a Cluster Tube System (such as Corning catalog # 4410) and acquired manually. The cluster tube can then be transferred to a regular FACS tube for acquisition.
Make sure that enough cells have been prepared for the staining. If there are not enough cells, you may choose to divide the staining into two separate experiments.
Depending on the application, the cell number needed for staining can be decreased. Successful staining has been done with1 x 105 cells/well.
Make sure to prepare cells for machine setup and compensation. These cells should be treated the same way as the cells for staining in the plates.
Handle the plates with care. Keep the plates upright at all times and be careful not to knock the plates over.
Protect the plates from exposure to light as much as possible.
Use extra caution when opening the plate cover (see Plate Preparation for instructions). After the cover is removed, do not move or apply any sealers on the plates until the cakes are reconstituted. Applying plate sealers or any cover onto the plate before the cakes are reconstituted can result in cakes being dislodged and stuck to the cover or escaping from the wells.
Some cell surface markers are sensitive to enzymatic digestion. If adherent cells are being used for staining, a mild enzyme or non-enzymatic dissociation buffer should be used when possible.
Make sure cells are in a single cell suspension. DNase treatment is recommended to avoid clumps caused by dead cells.
Acquire only 70 µL of the 160 µL total volume so that a second run can be performed if necessary.
Product Performance
The LEGENDScreen™ Human PE Kit was tested and compared with BioLegend's cataloged single vial liquid antibody reagents. For cell staining, human PBMCs were isolated and 3 × 105 cells were added to each well after the lyophilized antibodies were reconstituted. The cells were then stained for 20 minutes at 2 - 8°C, washed, and fixed with Fixation Buffer. The cells were then washed, resuspended in 160 µL of Cell Staining Buffer, and analyzed using a Beckton Dickenson FACS Canto II Flow Cytometry Analyzer.
The staining patterns and median fluorescence intensity between the lyophilized product and liquid antibodies are similar. Below are some representative data obtained when comparing the LEGENDScreen™ Human (PE) Kit vs. equivalent liquid antibodies.
Representative Data
Plate Maps
Below are the maps for the 4 plates provided with the LEGENDScreen™ PE Human Antibody Panel. To view the antibody in each plate, mouse over the wells. Click to find out more information on the specificity and antibody in each well.
One hour before the staining, perform the following steps to prepare the plates:
Remove the lyophilized plates from the aluminum pouches.
Centrifuge the plates at 600 X g for 5 minutes.
Check and make sure the lyophilized cakes settled to the bottom of the plates. Keep the plates upright at all times from this point forward. Handle plates with care so that the cakes are not agitated at any time.
Gently settle the plates on the bench. Hold the plate firmly and carefully remove the plate cover from one corner, one well at a time, for easy opening and preventing cross-well contamination. You may need to wiggle the cover a little while pulling back slowly.
Discard the cover. Do not reseal with the original cover or apply any sealer until the lyophilized antibodies have been reconstituted.
Reconstitute the lyophilized antibodies immediately, with 25 µL/well of deionized water using a multichannel pipette. To avoid crosscontamination, do not let the pipette tips touch the wells. Make sure that all the cakes are dissolved in water. If a cake is stuck on the side of the well, pipette the 25 µL water added to the well and rinse the cake down to the bottom.
Seal the plates with the plate sealers provided.
Wait at least 15 minutes before the staining procedure. Keep the plates in the dark.
Note:
PE-conjugated antibodies are light sensitive. Try to minimize the exposure of the plates to light as much as practically possible.
Do not open the pouches until the day you are ready to run the experiment. Once the plates are removed from the pouches, the antibodies must be reconstituted immediately.
If an experiment is not performed after reconstitution, plates can be sealed and stored in the dark at 4°C for up to one month.
Preparation of Cells
Obtain desired tissue (e.g. spleen, lymph node, thymus, bone marrow) and prepare a single cell suspension. Wash cells in 1X PBS or cell culture medium of choice, and resuspend in Cell Staining Buffer at a density between 4 × 106 and 1 × 107 cells/mL.
For cultured cells in suspension, spin and resuspend cells in Cell Staining Buffer at a density between 4 × 106 and 1 × 107 cells/mL.
For cultured adherent cells, dissociate cells using a mild enzyme (e.g. Accutase® Cell Detachment Solution, Cat. No. 423201) or non-enzymatic dissociation buffer (Cell Dissociation Buffer, enzyme-free, PBS, Cat. No. 13151-014, Life Technologies). Wash cells in 1X PBS or cell culture medium of choice, and resuspend in Cell Staining Buffer at a density between 4 × 106 and 1 × 107 cells/mL.
Filter the cells through a 40 µm cell strainer to remove any clumps. Keep the cells on ice before use. Approximately 22.5 mL of cells at a density between 4 × 106 and 1 × 107 cells/mL is needed for all three plates. Lower cell density (e.g. 1.5 x 106 cells/mL) might be used depending on the application.
Optional: Optional: Reagents that block Fc receptors (TruStain fcX™ (anti-mouse CD16/32) Antibody, Cat. No. 101320, BioLegend) may be useful for reducing nonspecific immunofluorescent staining.
Staining Procedure
Using a multichannel pipette, add 75 µL/well of cells (~ 3 - 7.5x 105 cells/well) to the plates as prepared above.
Set up extra tubes to stain cells for flow cytometer setup and compensation, if needed.
Using the multichannel pipette, gently mix the cell suspensions by pipetting up and down 2 - 3 times. Be sure to change tips between each row. Avoid creating bubbles while pipetting.
Incubate for 20 - 30 minutes at 4°C in the dark.
Spin the plates at 500 × g for 6 minutes to pellet cells in each well. Immediately after centrifugation, dump the supernatant into the sink by quickly inverting and flicking the plate. Gently blot the plate on a paper towel, being careful not to disturb the cell pellet.
Use a multi-channel pipette to deliver 200 µL of Cell Staining Buffer to each well. Gently mix up and down to resuspend cells. Be sure to change tips between each row.
Repeat step 5.
To fix the cells, using a multichannel pipette, aliquot 100 µL of Fixation Buffer into each well. Gently mix up and down to resuspend cells. Be sure to change tips between each row.
Incubate for 10 minutes at room temperature in the dark.
Repeat steps 5-6.
Repeat step 5 one more time.
Resuspend cells completely in 160 µL of Cell Staining Buffer per well and analyze using a flow cytometer. We recommend analyzing 70 µL of samples and collecting at least 5,000 events, but the end user may need to determine the optimal number of events to be collected. While the first plate is being analyzed, store the other plates at 4°C in the dark.
Tips for Successful Staining
Read the entire manual carefully before the experiment.
Plan the experiement in advance. Designate a full day for this experiment. Do not rush any step.
Make sure that the flow cytometer's autosampler is well maintained and working well before the experiment. If the flow cytometer does not have an autosampler, the samples from each well of the plates should be transferred to individual FACS tubes and read manually. In this case, the volume of the samples may need to be increased to avoid running dry.
Make sure that enough cells have been prepared for the staining. If there are not enough cells, you may choose to separate the staining into two separate experiments (e.g. stain two plates each time).
Depending on the application, the cell number needed for staining can be decreased. Successful staining has been done with 1 x 105 cells/well.
Make sure to prepare cells for machine set up and compensation. These cells should be treated the same way as the cells for staining in the plates.
Handle the plates with care. Keep the plates upright at all times and be careful not to knock the plates over.
Protect the plates from exposure to light as much as possible.
Use extra caution when opening the plate cover (see Plate Preparation for instructions). After the cover is removed, do not apply any sealers on the plates until the cakes are reconstituted. Applying plate sealers or any cover onto the plate before the cakes are reconstituted can result in cakes being dislodged and stuck to the cover or escaping from the wells.
Some cell surface markers are sensitive to enzymatic digestion. If adherent cells are being used for staining, a mild enzyme or non-enzymatic dissociation buffer should be used when possible.
Make sure cells are in a single cell suspension. DNase treatment is recommended to avoid clumps caused by dead cells.
Analyze only 70 µL of the 160 µL samples so that a second run can be performed if necessary.
Product Performance
The LEGENDScreen™ Mouse PE Kit was tested and compared with BioLegend's cataloged single vial liquid antibody reagents. For cell staining, cells were isolated from spleen and lymph nodes, and 3 × 105 cells were added to each well after the lyophilized antibodies were reconstituted. The cells were then stained for 20 minutes at 4°C, washed, and fixed with Fixation Buffer. The cells were then washed, resuspended in 160 µL of Cell Staining Buffer, and analyzed using an iCyt Eclipse Flow Cytometry Analyzer.
There were no significant differences in the staining patterns or median fluorescence intensity between the lyophilized product and liquid antibodies. Below are some representative data obtained when comparing the LEGENDScreen™ Mouse PE Kit vs. cataloged liquid antibodies.
Representative Data
Download the product manual to see more data comparisons.
Plate Maps
Below are the maps for the 3 plates provided with the LEGENDScreen™ PE Mouse Antibody Panel. To view the antibody in each plate, mouse over the wells. Click to find out more information on the specificity and antibody in each well.
