BioLegend Web Updates
Blog - It’s Not All About the Antibodies


The irreproducibility of published data has become a legitimate concern in recent times. In 2012, a landmark study conducted by Glenn Begley and his team concluded that the majority of cancer research studies were not reproducible, demonstrating that a startling 47 out of 53 published findings could not be replicated (Nature 483, 531-533; 2012). The implications for biomedical research are potentially staggering considering the billions spent on research every year, with some suggesting that as much as $28 billion is wasted on irreproducible pre-clinical research every year (Freedman LP, et. al. PLoS Biology 13: e1002165). Antibodies used in research have become a popular target for blame. Many instances of poor specificity, cross-reactivity, and lot-to-lot variability have cost researchers millions and possibly billions, and will likely continue to do so in the future. But, we need to have a keen eye for other factors that are likely to contribute to experimental irreproducibility beyond just the antibodies.
Bias

As humans, we all have preferences, desires, pressures, and feelings that might be reflected in the experimental outcomes, no matter how hard we try to separate them from data. One of the difficulties with a hypothesis-based approach is that most researchers want the data to support their original hypothesis. This inherently introduces bias from the start. Along the way, bias contributes to almost all aspects of experimentation. Below is a list of some areas, but not all, where bias has effects:
  • Selective reporting, i.e. excluding outlier data
  • Data selecting, i.e. counting phenotypes by microscopy
  • Limited data sets
  • Interpretation of data
  • Experimental design
  • Reagent selection
  • Collaborators
There are a few straightforward steps that one might take to try to remove bias as much as possible. These might include:
  • Blinding and randomization of treated versus control groups
  • Including as many controls as possible, both positive and negative
  • Use proper statistics to determine significance
  • Use well published protocols and reagents
  • Use quantifiable measurements, making sure all instruments and assays are properly calibrated


Comic from Dzu-Doodles.

Variability

Commercial resources for critical research reagents like cell culture media, antibodies, and recombinant proteins may go through many rounds of validation testing prior to sale, but what about other day to day materials that are commonly used? Some things to consider:
  • Do you routinely check the quality of your DI water?
  • Are any of your chemicals expired?
  • When was the last time your instruments and equipment were calibrated?
  • What is the range of temperature fluctuation in your refrigerator?
  • Do you always buy the same reagents from the same source? Even if you do, how does the supplier control lot-to-lot variability?
  • How long do you keep stock solutions? Would you know if they are contaminated?
  • Are conditions for the storage and handling of mice consistent from week to week, and month-to-month?
  • Are your cell lines authenticated? Are they mycoplasma free?


Comic from Dzu-Doodles.

The potential for variability and irreproducibility, even within the same lab, are likely too high if processes, reagents, equipment, and protocols are not consistent.


In addition to variability in research materials, there is also an inherent variability in human and research animal subjects that makes reproducibility a challenge.


Trainees


The academic system relies on a large proportion of its data to be generated by people regarded as "trainees," either graduate students or post-docs, while experienced Principal Investigators spend their time seeking funding and writing grants. Their status as trainees reflects on their lack of expertise in a subject matter or experimentation, often times both. Yet, the expectation is that they perform the majority of the experiments to generate data for publications. Even in labs with the most well renowned PI's, there will always be new trainees entering, and creating new data. In addition to this, experimental training comes in vastly different forms and quality. Some students are trained by experienced mentors, while others may be trained by the student who started in the lab last quarter. The lack of universal standards for training should be major concern for reproducibility in science.



Comic from Dzu-Doodles.



Conclusion


It's not all doom and gloom though. Scientific discoveries have led to multitudes of breakthroughs that have directly impacted the quality and quantity of life we get to have today. New discoveries will be built upon the groundwork of science that came before. Despite all the imperfections in the way we do science, the body of work that science builds upon provides us a means of constantly changing our understanding of the world and ourselves. We do have a long way to go in reducing irreproducibility, but just consider: it's not always the antibodies.


Comic from Dzu-Doodles.

Learn how BioLegend addresses Validation and Reproducibility.
Written by Dzung Nguyen, PhD.


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Video - Isolate PBMCs with Lymphopure™
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Blog - Increase Your Knowledge: Learn how BioLegend helps you stretch your neuro-dollar further.



Know what you’re working with
Reproducibility is the cornerstone of science. Using a consistent amount of antibody from experiment to experiment is crucial to ensure the reproducibility of your data. BioLegend offers three sizing options: microgram, microliter, and test sizes. Understanding the differences between the formats can help you decide which format will best suit your needs. Microgram sizing is perhaps the best way to know exactly how much antibody you are buying, and is often the best value. Similarly, microliter sizes are sold at a set volume with the concentration clearly stated on the TDS. Both the microgram and microliter sizing options are best suited for optimizing titration, and allow for consistent antibody usage across experiments. They also make it easier to calculate how much isotype control to use. Test sizes are offered as a convenience, where the titering has already been done for you. Test size antibodies are meant to be used at a set volume (typically 5 μL/test).




Discover more, for less
Technical and biological replicates ensure sound and reproducible results, but come at the cost of increased reagent usage. BioLegend offers competitive pricing on our neuroscience products, so you get more replicates and more data. Check out the table below to see how you can get more value in your vial when buying the same antibody clone from BioLegend and a few of our competitors. For ease of comparison, we converted the size of those products sold in a 100 μL format (marked with an asterisk) to total μg per vial based on their concentration of 1 mg/mL.



Description Clone BioLegend
(total μg/vial)
Competitor A Competitor B
α-Synuclein Phospho
(Ser129)
P-Syn/81A 100 μg*- $285 50 μl- $419 100 μg- $333
Brevican N294A/6 100 µg*- $140   100 µg- $353
GluR2 L21/32 100 μg*- $140 100 μg- $419 100 μg- $353
GRK N145/20 100 μg*- $140 100 μg- $405 100 μg- $353
KCC2 N1/12 100 μg*- $140 100 μg- $409 100 μg- $353
Laforin N84/37 100 μg- $140 100 μg- $389 100 μg- $343
MECP2 N227/21 100 μg*- $180   100 μg- $343
NeuN 1B7 100 μg*- $202 100 μg- $429  
Pan-shank N23B/49 100 μg- $140 100 μg- $409 100 μg- $353
VDAC1 N152B/23 100 μg*- $140 100 μg- $405 100 μg- $343


One Product, Multiple Applications

Here at BioLegend, we quality test and validate our products across a wide range of applications. Many of our reagents pull double, triple, or quadruple duty, meaning you can perform more assays with the same reagent. These applications are listed on the TDS so you know what your shiny new antibody can do.
Purified anti-Tubulin β 3 (TUBB3) Clone TUJ1




Particular application not listed? Even if we don’t test a clone for a particular assay in-house, our dedicated team of technical support scientists scour the literature for other possible applications for our clones so you don’t have to.



Generous Pricing and Discounts
You can learn more about our neuroscience products with our helpful webpage. For discounts, quotes, and help with our products, get in touch with our technical application scientists.

Contributed by Christopher Dougher, PhD.


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Blog - 3 Considerations for Intracellular Flow Cytometry (ICFC)
Designing an experiment for staining surface markers is pretty straight forward. For the most part, there are not specific buffer requirements when surface staining, and negative controls are usually restricted to unstained samples and isotype controls. If your area of research leads you to explore intracellular targets, there are additional things to consider before moving forward with your experiment.


The Matrix. Village Roadshow Pictures.

1. Choosing the appropriate concentration.
While using the optimal concentration in surface staining is important, doing so when performing intracellular staining is critical. The suggested amount of antibody for use in your flow experiment can be found on the Technical Data Sheets under 'Recommended Usage'.


The recommended concentrations provided by BioLegend are determined by performing 3-5 point titrations and choosing the results that have the best signal to noise ratio.

While these concentrations provide a good starting point, there may be situations in which you will need to titer your antibodies to find the optimal concentration for your sample type. If you use too little antibody, you run the risk of not detecting a signal or not being able to distinguish between your negative and positive populations. If you use too much antibody, you could potentially start to see increased background staining that can lead to an entire cell population shifting, thereby leading to incorrect analysis of your data. This phenomenon is known as "intracellular shift".



2. Choosing the appropriate buffer system.
Staining for intracellular targets requires one to fix and then permeabilize the cells. The most common fixative used is paraformaldehyde (PFA) which stabilizes the cell membrane prior to permeabilization with a detergent or alcohol, allowing the antibodies directed against intracellular antigens to enter the cell and bind to their target. The location of your target of interest will determine the most appropriate buffer system for your experiment.
Cytoplasmic Targets

Staining for cytoplasmic targets, such as cytokines, requires a two-step fixation and permeabilization protocol. For this, we offer two different options:

  1. Fixation Buffer and Intracellular Staining Permeabilization Wash Buffer (10X).
  2. Newly released Cyto-Fast™ Fix/Perm Buffer Set.


While the fixation and permeabilization buffers (10X) are sold as separate products, our new Cyto-Fast™ Fix/Perm Buffer Set provides a fix/perm solution and perm wash solution (10X) that have been QC tested together as a set and they often improve the signal-to-noise ratio as compared to our traditional fixation and permeabilization buffers. This provides more consistent and reproducible results for your intracellular staining experiments.
Nuclear Targets


Staining for nuclear targets, such as transcription factors, requires a buffer system that will not only permeabilize the outer cell membrane, but also the nuclear membrane. In order to obtain optimal staining of transcription factors and other nuclear targets, we recommend using our True-Nuclear™ Transcription Buffer Set. This buffer is an improved version of our FOXP3 Fix/Perm Buffer Set. Some nuclear permeabilization buffers can have a detrimental effect on tandem dyes, thereby leading to a loss of FRET and increased compensation into the tandem dyes donor channel.


Whole blood was stained with human CD3 APC/Cy7 (blue and red) or APC/Fire™ 750 (green and purple), then lysed, washed and fixed under the following conditions: A) Fixed with FOXP3 Fix/Perm Buffer followed by permeabilization with FOXP3 Perm Buffer (purple and red). B) Fixed with True-Nuclear™ Fix Buffer, then permeabilized with True-Nuclear™ Perm Buffer (green and blue). Compensation requirements between APC and APC/Cy7 channels increased significantly for the FOXP3 Fix/Perm Buffer Set versus the True-Nuclear™ Transcription Factor Buffer set for both APC/Cy7 and APC/Fire™ 750.

Phosphorylated Targets


The use of phospho-specific antibodies for the analysis of phosphorylated proteins requires a buffer system that is a little harsher than the ones used for cytoplasmic and nuclear targets. We recommend staining phosphorylated proteins with our True-Phos™ Perm Buffer, which is methanol based, and specifically formulated for the detection of these phosphorylated proteins.


Human whole blood was stimulated with (right) or without (left) IL-6 for 15 minutes, then treated with RBC/Lysis Fixation Solution and permeabilized with True-Phos™ Perm Buffer, then stained with CD3 APC and STAT3 pY705 (clone 13A3-1) FITC.

Due to the fact that the True-Phos™ Perm Buffer is methanol based, special considerations need to be given to the choice of any antibodies used for surface staining. Methanol can alter or destroy the epitope that is recognized by an antibody, so it is good practice to do a small scale pilot study to determine if any of your surface stain antibodies can recognize a fixed epitope. You can also check our fixation page to see if we have tested a particular clone already. Please note, the clones on this page were evaluated using 4% PFA as the fixative, but it is a good source of information to give you an idea of whether your clone might stain a fixed epitope. If any of the clones you are working with do not recognize a fixed epitope, you can stain prior to using the True-Phos™ Perm Buffer if the conjugated fluorochrome is methanol resistant.



"Special Consideration" Targets

There are some clones that march to the beat of their own drum so to speak, and in order to achieve optimal staining results, require their own intracellular staining protocol using buffers we haven’t covered yet. One example is our Ki-67 clones, Ki-67 and 16A8. For optimal staining, these clones require the use of ice cold 70% ethanol as a combined fixative-permeabilizing agent, and their specific protocol is outlined on the TDS.


The moral of the story is to ensure you have chosen the appropriate buffer system for the intracellular targets you are looking to evaluate.
3. Choosing the appropriate controls.

The use of the appropriate controls in your experiment is critical for being able to analyze your data correctly. You can learn more about controls with our Flow Controls webpage. 
Isotype Controls

While isotype controls are a good choice when your experiments are limited to surface staining, it is generally not recommended they be used as the only control when performing intracellular staining. Occasionally when performing intracellular staining you can see shifting of the whole population, which can lead to incorrect analysis/assumption of what is actually staining. This is why isotype controls are generally used as a guide, and ideally should not be used as the ultimate criteria for setting the negative population, especially when there are clear cut positive and negative cell populations. 
If you set the quadrants based on isotype control (black line), it appears as if you have more signal in Q1 and Q2. If you set the quadrants based on the inherent negative and positive populations (red line), you get a more accurate assessment of the signal in Q1 and Q2. 

Biological Controls

When staining with antibodies directed against cytokines or cell signaling molecules, the use of a biological control is a good choice. For example, if your samples include cells that have been stimulated with PMA & Ionomycin, LPS, or CD3/CD28, you should also include the same cell type that has not been stimulated. This is important for understanding the difference between the basal level of expression and any increase in expression due to upregulation caused by your stimulatory conditions.


Th2 polarized human peripheral blood lymphocytes were treated with monensin alone (left) or treated with monensin and stimulated with PMA and Ionomycin (right) for three hours. Cells were then surface stained with CD3 (X axis) and intracellularly stained with GM-CSF (Y axis).

Fluorescence Minus One (FMO) Controls

Another important control to include in your intracellular staining experiment is a fluorescence minus one, or FMO. The FMO control consists of staining your samples with all but one of the conjugates in your panel. This control is not only useful when you have targets expressed at low levels, but also when it is difficult to distinguish between your negative and positive populations and to account for any spectral overlap you may encounter between fluorochromes in larger panels.



After reading this blog, we hope you have a good understanding of what is necessary when designing and performing an intracellular staining flow cytometry experiment. Choosing the optimal antibody concentration, correct buffer system, and best control(s) will ultimately lead you to generating consistent and reliable data. Should you have any additional questions regarding your intracellular staining experiment, do not hesitate to contact our tech support group at tech@biolegend.com.


Contributed by Kellie Johnson.


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Podcast Episode - The Story of Henrietta Lacks
In our return to the podcast, we discuss the first immortal cell line, HeLa cells, and the controversial circumstances of how they were obtained from Henrietta Lacks.

Topics

The Controversy Over HeLa Cells
The Henrietta Lacks Foundation
The Legacy of Henrietta Lacks (Johns Hopkins Medicine)
Johns Hopkins to name research building after Henrietta Lacks
Moore v. Regents of the University of California
Dat Tran and Lick-A-Bubble
Harry Potter Spoilers (15:32-15:50)

Keywords: Henrietta Lacks, podcast, HeLa cells, cell line, telomeres, telomerase, polio, senescence, Helen Lane, cancer, Johns Hopkins, George Otto Gey, The Immortal Life of Henrietta Lacks, Rebecca Skloot, John Moore, David Golde


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Video - Aβ Aggregate Abs, Customer Testimonial
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Blog - Answers to 5 Common Questions about the Activity of Recombinant Proteins
Many of you often use recombinant proteins to address scientific questions related to cell function and behavior in different research models. However, you may still be puzzled about what the terms specific activity, ED50, and International Units (IU) mean and how these measures correlate with each other. In this blog, we will touch base on the fundamentals of protein biological activity and discuss the most common questions about this topic.

1. How is protein biological activity measured?


The biological activity of recombinant proteins is usually provided in units per mass (μg or mg) and is routinely measured using a bioassay. The range of assays that can be used to measure the biological activity of proteins include cell proliferation assays, cytotoxic assays, binding assays, ELISAs, and chemotaxis. Figure 1 represents a few methods we use in-house to measure the biological activity of our recombinant proteins.

In order to calculate the specific activity of a protein you need to get the ED50. The ED50 represents a protein concentration measured in ng/ml required to elicit a 50% maximum response in a specific bioassay. This method of expressing activity should only be used for proteins whose dose-response curves are sigmoidal in shape. The following formula can be used to transform the ED50 to specific activity: Specific Activity (Units/mg) = 1 x 106 / ED50 (ng/ml)




A. Cytotoxic effect of mouse TNFα on the L929 mouse fibroblast cell line in the presence of actinomycin D measured by Deep Blue Cell Viability Kit. B. Cell adhesion of Calcein-AM labeled Jurkat cells to immobilized human Galectin-9.




C. RBC agglutination in the presence of human Galectin-9. D. Inhibition of mouse TNFα production by mouse TRANCE.


Figure 1. Examples of in-house assays used by BioLegend to measure the biological activity of our recombinant proteins



2. Can the ED50 calculated in the assay A be used in assay B?


No. The specific activity of a certain protein may differ depending on the assay and cell type used. For example, Recombinant Human IL-2 can activate proliferation of IL-2 dependent cell lines, CTLL-2 and HT-2. The same lot of recombinant protein provides different ED50 values depending on the cell line used for stimulation. In the CTLL-2 stimulation assay, the ED50 is 0.26 ng/ml corresponding to a specific activity of 3.73 x106 units/mg. Because the HT-2 cell line is more sensitive to IL-2 proliferation, the ED50 in the dose-dependent stimulation of HT-2 cells is 0.6 ng/ml, corresponding to a specific activity of 1.65x 106 units/mg (Figure 2).

Since our products are used for many different purposes, it is impossible to provide the ED50 for every possible experimental scenario. Our standard bioassay is used to confirm the biological activity of the protein. Therefore, we recommend that the end-user test each lot of the protein and each new set of experimental conditions in a dose response experiment to determine the concentrations that work best for their specific assays.
Figure 2. Dose-dependent proliferation of CTLL-2 and HT-2 cell lines in response to human IL-2
3. Why is the ED50 provided as a range?


You may have noticed that the Technical Data Sheet of our recombinant proteins provide the range for ED50 and specific activity. Why? We have stopped issuing the exact specific activity values for each lot of recombinant protein, in part, 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, cell passage number, culture media, and end-user technique. Given these variables, providing an exact activity value may be misleading.


4. Is the specific activity reported in units/mg is the same as the International Units (IU/mg)?


International units are assigned to international standards provided by the World Health Organisation (WHO). International standards are manufactured according to WHO guidelines and serve as a 'gold standards' that can be used by different manufacturers or end-users to calibrate their own products.

Our experimental ED50 and specific activities are not directly related to or convertible to International Units (UI or IUs), as our testing procedure does not follow the International Standard set by the WHO. The best way to determine how the specific activity of our reagents converts to IUs would be to acquire a known IU reagent and perform an experiment side-by-side with our recombinant protein to see how they compare.

5. Can the recombinant proteins from different vendors be compared using size or units?


As mentioned earlier, recombinants derived from different vendors may have different activity per μg or mg size. Different suppliers may have their own method of defining a unit. In order to compare the biological activity of proteins from different vendors, their activity should be compared side-by-side in a titration experiment using the same bioassay.

Hopefully we were able to clear up any confusion related to the biological activity of recombinant proteins. If you still have questions related to this topic please contact us at tech@biolegend.com.


Contributed by Ekaterina Zvezdova, PhD.


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Video - Characterizing Antibody S16007D for P2RY12 Detection in Microglia
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Blog - Using Flow Cytometry in Neuroscience Research

Sample Preparation


One of the initial hurdles in establishing flow cytometry applications in neuroscience was preparing suitable samples. When using whole brain tissue, generation of single cell suspensions is possible through a variety of methods.






Mechanical Dissociation

First, tissues need to be mechanically dissociated into small fragments using a razor blade prior to processing.


Enzymatic Digestion

Enzymes such as Trypsin, Accutase®, or Papain can be used to digest whole brain tissue samples.
Density Gradient

Percoll® or sucrose gradients help to remove myelin from the sample. For this, we use a 70/37/30% Percoll® gradient.


Cell Markers

Flow cytometry in neuroscience is useful when you need to study or isolate a homogenous population from a heterogeneous sample, such as primary cell suspensions or in vitro differentiation of neural stem cell cultures. Identification of cell type-specific markers allows for detection or isolation of these cells through magnetic cell separation or cell sorting. Cell type-specific markers have been identified for a variety of neural cell types including astrocytes, microglia, and oligodendrocytes.






CD56 (NCAM)
CD24
CD200
CD11b
CD45
CX3CR1
P2RY12
TMEM119
GFAP
GLAST




A2B5
PDGFRα
NG2
CD15
CD29
CD133
CD184
CD171
Nestin
Notch-1
Notch-2
Sox-2

Check out these references using flow cytometry to identify neural cell types:




Organelle Markers & Function


Altered organelle morphology and health is involved in numerous neurodegenerative diseases. For example, mitochondrial dysfunction and accumulation of reactive oxygen species is common in Alzheimer's and Parkinson's disease. When performing flow cytometry, in conjunction with cell type-specific markers, you can include a cell-permeant dye like MitoSpy™ as an indicator of cell health and mitochondrial function.

Similarly, lysosomal proteins help clear aggregated proteins associated with disease. Antibodies against lysosome markers like LAMP-1 or LAMP-2 can be used in flow cytometry to compare lysosome function in normal and disease states.

Find your antibodies for Neuroscience and Flow Cytometry at: biolegend.com/neuroscience
Contributed by Kelsey Swartz, PhD.


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Video - FFPE Tissue Slide Preparation and Processing
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Video - Immunofluorescence Tissue Staining
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Video - Chromogenic Tissue Staining using DAB Detection System
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Video - Frozen Tissue Slide Preparation and Processing
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