As important as good data and results are, having the right controls to interpret and troubleshoot your experiment is just as critical. Whether you need to gate your populations accurately or identify whether your result is unique or artifactual, controls provide context and clarity. Click on each subject below to learn more about the proper control reagents BioLegend offers and for which scenario they can optimally be applied to.

Fc Blocking Controls...

Block the non-specific detection of the Fc component of all antibodies. It is most appropriate for samples where the cells express Fc receptors that can exhibit non-specific binding of antibody.

Antibodies consist of two heavy chains (blue) and two light chains (red) connected by disulfide bonds. The antigen binding region consists of highly variable regions that allow antibodies to recognize one target out of approximately ten billion (based on recombination events). Beyond these regions, the sequences of antibodies tend to remain relatively constant. Experiments in the 1960s utilized enzymes like papain to help understand the structure of antibodies. Papain is a thiol-endopeptidase that cleaves peptide bonds within the hinge region, creating three fragments: two were found to be identical and named Fab for their ability to bind antigen; the remaining fragment was named Fc for its tendency to crystallize during cold storage.

image

An antibody has two main functions: to bind foreign antigens and to mediate effector functions of other immune pathways. The latter can be accomplished with the help of Fc receptors, which are present on many cell types, including granulocytes, B cells, macrophages, and dendritic cells. As these Fc receptors recognize the Fc portion of an antibody (and potentially any pathogens attached to the Fab region of the attached antibody), this mechanism triggers several downstream effects, often including phagocytosis, activation or antibody-dependent cell-mediated cytotoxicity. This is commonly associated with Fcγ receptors, which recognize IgG antibodies. Fcε receptors can help launch allergic responses and degranulation.

As these receptors have a propensity to bind the Fc portion of antibodies, they can present false positives in your analysis. To prevent this background staining, we recommend using Human TruStain FcX™ or Mouse TruStain FcX™ PLUS.


Mouse TruStain FcX™ PLUS is an antibody (clone S17011E) specifically directed against CD16 and CD32 (FcγRIII and FcRγII respectively) via the Fab portion of the antibody, with improved Fc blocking capabilities compared to the original mouse TruStain fcX™ (clone 93). Simultaneous detection of CD16 and CD32 with mouse TruStain FcX™ will depend on the clones used and whether the same epitopes are recognized by these reagents. Clone S17011E blocks both clone 93 and 2.4G2, which are also raised against mouse CD16/32.

Human TruStain FcX™, on the other hand, is a proprietary blend of specialized human IgG immunoglobulins that bind to Fc receptors via the Fc portion of the immunoglobulin. Despite occupying these Fc receptors, Human TruStain FcX™ is still compatible with flow cytometric staining with anti-Fc receptor antibodies, such as anti-human CD16 (clone 3G8), CD32 (clone FUN-2), and CD64 (clone 10.1) antibodies.

human trustain image

Human TruStain FcX™ treated (filled histograms) or non-treated (open histograms) THP-1 cells stained with anti-human HLA-DR PE antibody (red) or an isotype control (IgG2a PE, blue). Non-treated cells show false-negative HLA-DR staining due to high binding of mouse IgG2a isotype.

If you are using a secondary antibody that might detect the isotype of either mouse or human TruStain, you might seek out an alternative. Fc blocking using serum originating from the same host species as the labeled antibody is one possible method. For example, if you are using a labeled secondary antibody that is of a rat isotype, you could use rat serum to block non-specific binding of immunoglobulins/antibodies to the Fc receptors.

Monocyte Blocking...

Prevents live monocytes from non-specifically binding to fluorophores.

Monocytes and macrophages can non-specifically bind to fluorophore-conjugated antibodies used in cell surface staining of live cells. While there are some publications which propose this phenomenon may occur due to the function of CD64, Fc blocking does not ameliorate this effect. This effect is also not cyanine-specific. Our testing shows that PE/Dazzle™ 594, which is not a cyanine-based acceptor fluorophore, can exhibit non-specific binding to monocytes. On the other hand, cyanine-based fluorophores like Alexa Fluor® 647 and the cyanine-based acceptors of some of the Brilliant Violet™ tandems do not exhibit non-specific binding to monocytes. Our testing has shown this effect appears to be quite selective for any PE, PerCP and APC tandems. (please note that the tandem fluorophores of competitors were not included in our analysis).

 

BioLegend has now formulated an effective blocking reagent, True-Stain Monocyte Blocker™. It is a non-antibody based blocking solution that has been shown to reduce non-specific monocyte binding due to the fluorophore and does not affect the desirable specific staining of monocytes. To learn more about which fluorophores might require True-Stain Monocyte Blocker™, take a look at the chart to the right. To learn more about this reagent, visit our webpage.

Fluorophore conjugates recommended for use with True-Stain Monocyte Blocker™ Fluorophore conjugates that likely do not need True-Stain Monocyte Blocker™
PE/Cyanine5 Non-tandems: FITC, PE, APC, Pacific Blue™, PerCP, BV421™, BV510™, Alexa Fluor® dyes, Spark Dyes
PE/Cyanine7
PE/Dazzle™ 594
Fire Dyes Brilliant Violet™ tandems: BV570™, BV605™, BV650™, BV711™, BV750™, BV785™
APC/Cyanine7
PerCP/Cyanine5.5
panels

Human PBMCs were stained with 5 µl/test of antibody in 100 µl of cells at 1 x 106 cells/ml with (bottom row) or without (top row) True-Stain Monocyte Blocker™.

Isotype Controls...

Represent the non-specific binding present on cells due to non-specific binding of an antibody to the Fc component or conjugated fluorophore of the antibody. 

Even after blocking Fc receptors, it can be helpful to include an isotype control to see how much non-specific binding your cells may exhibit. For instance, certain cyanine dyes also can bind to cells independent of Fc receptors. In these scenarios, isotype controls would also help indicate non-specific binding.

An isotype control matches the constant region of the antibody to the antibody you plan on testing. However, the Fab portion of an isotype control is raised to be low affinity and against a target that is not likely to be found on the cell type being analyzed (i.e., Dinitrophenyl or Keyhole Limpet Hemocyanin). Essentially, if an isotype control binds to your samples, it’s through the Fc region. This helps tell you how much of the stain of your test antibody (which possesses the same constant or Fc region as the isotype control) is due to the Fc region of the antibody being recognized non-specifically.

You should also make sure to use the same amount of test and isotype control antibody. If the antibody is provided in test size format, identify the microgram quantity of the antibody for that volume and obtain an equal mass of the isotype control. Avoid purchasing isotypes and test antibodies from different companies, as the fluorophore:protein (F:P) ratio may not be consistent between different manufacturers.


View all of our isotype controls…

cd11c image

In this plot, a fully stained sample is shown (red) or a control sample where the isotype control was substituted for PE/Cyanine5 anti-human CD11c (blue) in an FMO. Notice the population in the green box shifts to the right on the x-axis when using an isotype control, indicating some non-specific binding. However, despite this background, the reagents are well-matched to still make distinct gating on the double positive population possible.

Human

Receptor Main ligand Affinity Cell Distribution
FcεRI IgE Very high Mast cells, Basophils, Monocytes, DCs, Neutrophils
FcεRII (CD23) IgE Low B cells, T cells, NK cells, DCs, Eosinophils, Macrophages
FcγRI (CD64) IgG1> IgG2, IgG3, IgG4 High Monocytes, DCs, Macrophages, Neutrophils , Eosinophils
FcγRIIA (CD32) IgG1 and IgG3>IgG2>IgG4 Low-Medium NK cells, DCs, Macrophages, Monocytes, Neutrophils, Platelets
FcγRIIB* IgG4>IgG1, IgG3>IgG2 Low-Medium Monocytes, DCs, Macrophages, B cells, Neutrophils, Basophils, Mast Cells
FcγRIIC IgG1>IgG3>IgG4>IgG2 Low-Medium NK cells
FcγRIIIA (CD16) IgG1>IgG2 and IgG3>IgG4 Low-Medium Macrophages, NK cells, γδ T cells, Monocytes, DCs
FcγRIIIB IgG1 and IgG3>IgG2 and IgG4 Low-Medium Neutrophils, Mast cells, Eosinophils
FcαRI (CD89) IgA1, IgA2 Low Monocytes, Macrophage and DC subsets, NK cells, Neutrophils, Eosinophils
Fcα/μR IgM>IgA High for IgM, medium for IgA Germinal B cells, Follicular DCs.

*Note that FcγRIIB is actually inhibitory and prevents cell activation. To determine if various cell lines express Fc receptors, you may have to check with the company you purchased it from or the literature.

Mouse

Receptor Main ligand Affinity Cell Distribution
FcεRI IgE Very high Mast cells, Basophils, Monocytes, DCs, Neutrophils
FcεRII (CD23) IgE Low B cells, T cells, NK cells, DCs, Eosinophils, Macrophages
FcγRI (CD64) IgG2a> IgG1, IgG2b, IgG3 High Monocytes, DCs, Macrophages, Neutrophils , Eosinophils
FcγRIIB* IgG1 and IgG2b>IgG2a Low Monocytes, Macrophages, B cells
FcγRIII (CD16) IgG2a and IgG2b>IgG1 Medium NK cells, Macrophages, γδ T cells, Monocyte subsets
FcγRIV IgG2a, IgG2b, and IgE Medium Monocytes, DCs, Neutrophils
Fcα/μR IgM>IgA High for IgM, medium for IgA B cells, Monocytes, and Macrophages

References:

  1. Pleass, R. J. 2011. Parasite Immunol. 31:529. Pubmed
  2. Smith, K.G. and Clatworthy, M.R. 2010. Nat. Rev. Immunol. 10:328. Pubmed

Fluorescence Minus One...

Are gating controls useful in large multicolor panels to give accuracy to the gate assignment.  FMOs are particularly useful in 10+ color assays and when the expression of a positive population is poorly defined from the negative population.

FMOs, or Fluorescence Minus One controls, can be extremely helpful, especially when your target antigen is expressed at low levels, or you are concerned with the amount of spectral spillover from other fluors in a large panel. As the name suggests, an FMO consists of the full panel of antibodies while leaving out the target antibody of interest. In some instances, an isotype control can be used in place of the test antibody, which can provide a more complete picture of background signal, from both non-specific binding and fluorescent spillover.

 

 

For additional information on FMOs and compensation, read through this publication by Mario Roederer.

 

 

Compensation controls...

Are a correction factor applied to each channel in use on a flow cytometer to correct for spectral spillover of unintended fluorophores into their neighboring channels.  Compensation controls aid in accurate gating strategies and the avoidance of artefactual events.

Compensation controls are typically necessary for any flow experiment. Fluorophores emit along a spectrum around the peak emission. As a bandpass filter will “catch” wavelengths within its range indiscriminately, compensation should be applied in order to correct for spectral overlap between fluorophores. Compensation controls are single color-stained controls. They can be prepared as stained cells, however, compensation beads are the most robust reagent to use for this purpose.

It should be noted that high compensation values are not inherently indicative of poor reagents. These values can be directly influenced by voltage changes. What’s more important is that the compensation applied is accurate so that the cell populations can be more easily visualized and gated upon.

The four standard practices listed below help to ensure an accurate calculation of compensation:

  1. The reagents applied to the compensation controls, and experimental samples must be from the same vial of reagent and ideally treated the same until acquired.
  2. The autofluorescence of the compensation control must be identical for both the negative and positive events.
  3. Each compensation control's positive population must be at least as bright as any positive signal in the experimental sample.
  4. To ensure precise compensation, collect enough events to estimate median fluorescence in all channels as well as the negative population.

Compensation Beads...

Are stained with single color reagents that present the machine with enough spectral information from the fluorophore in use to generate an accurate compensation matrix.   

Compensation Controls can be generated using single color stained cells or beads. There are limitations to utilizing cells as compensation controls. They must have enough expression of the antigen to generate enough data points for an accurate calculation of compensation for that fluorophore. There also needs to be enough cells in the sample to distribute amongst all of these different kinds of controls and still have enough for the experiment itself.

The alternative is to use compensation beads. Depending on the vendor, the beads will bind a variety of host isotypes and can be quickly mixed with the antibody of choice to create a compensation control. Single color stained cells are ideal for setting the voltages to be applied to the experiment to ensure the positive and negative populations are both within the ideal linear range of detection. Compensation beads are more accurate to populate enough data from the negative and positive populations for the compensation algorithm to calculate. 

 

 

Either compensation beads or single color controls can be suitable for use as compensation controls. The important factors are that there are enough positive and negative events as demonstrated in the histogram using beads. However, since the PMT voltages applied in the assay are determined based on the biological sample to be analyzed, they may not be optimal for very brightly stained compensation beads. In the histogram where beads are employed, the positive peak is still able to be gated on accurately, but is in danger of going beyond the detectable range. In the second example, when using single color stained cells expressing CD44, the cells in this instance will not be suitable since there are not enough negative cells. In addition, the large variance of the MFI+ population can add to additional error in compensation calculation.

Rainbow Particles...

Are useful for ensuring your flow cytometer’s lasers are properly calibrated and aligned. 

First and foremost, it should be noted that Rainbow Particles are not compensation beads, nor can they be used to estimate cell size/number. Rainbow Particles are designed to ensure a flow cytometer is properly calibrated, standardize instruments for longitudinal studies, and evaluate new instrument hardware (e.g., bandpass filters, photomultiplier tubes (PMTs) and lasers). They are excited by any wavelength in the range of 365-650 nm, which includes the violet (405 nm), blue (488 nm), yellow/green (561 nm), and red (633 nm) lasers. They emit in all channels off of those lasers, but at varying intensities.  Rainbow beads can be found in 1 peak, 6 peak and 8 peak options and help characterize the range of sensitivity and linearity of the instrument PMTs and lasers. Rainbow Particles are not compatible with the UV laser.


When you excite fluorophores conjugated to antibodies bound to your cell, you want to ensure that they fall within an ideal range of performance for each laser’s PMT positions. This can be monitored by using the particles and plotting a channel from one laser against a channel from the other laser.

 

 

In the “bad” alignment plot, the violet laser may be underpowered or improperly aligned.

 

As mentioned previously, we offer one, six, and eight peak options, each of which can aid with longitudinal studies to check whether peaks routinely show up at the same fluorescent intensity using a calculation of staining index. For the one peak choices, there are mid-range and  bright intensity peaks. The mid-range option can help represent antigens of moderate expression, while the bright intensity option is ideal for highly expressed targets. Six or eight peak options offer a larger variety of fluorescent intensities. While six peaks is typically enough for most users, the eight peak option can be selected if more sensitivity is desired or the linear range of the PMT or photodiode is expanded.

For more information on the use of calibration beads, take a look at this article by Perfetto, et al.

 

Precision Count Beads™…

Are useful for counting absolute cell numbers in a sample of varying volume.

Precision Count Beads™ are designed for counting the absolute number of cells in a complex population and other particles by flow cytometry. The cell count in the original sample can then be back-calculated. Very few flow cytometers are capable this function, so Precision Count Beads™ are helpful for users without this function built into their cytometer.

Precision Count Beads™ are excited by the violet (405nm), blue (488nm), yellow/green (561nm), and red (633nm) lasers, and emit between 400-800 nm. As the beads have a low forward scatter and high side scatter, this must be taken into account when adjusting the voltage of the scatter channels to ensure the beads are on scale. Vortexing the beads prior to use and using “reverse pipetting” are important for delivering aggregate-free, well distributed accurate amounts of beads.

Precision Count Beads™ can work with all kinds of assays including a lyse, no-wash whole blood assay.

 

 

As shown in the top left plot, Precision Count Beads™ have a very high 
side scatter profile, so the voltage may need to be adjusted accordingly.

 

Autofluorescence Controls...

Are most appropriate when your samples may exhibit naturally high levels of autofluorescence that may differentially impact the sensitivity of resolution in each detection channel.

Different cell types and tissues have varying levels of inherent fluorescence. Major sources of autofluorescence include NADH, riboflavin, metabolic cofactors, the crosslinking of primary amines by paraformaldehyde, and certain biological structures (e.g., mitochondria, lysosomes). These proteins and molecules are more easily excited at lower wavelengths (i.e. from the UV, violet, and blue lasers) and will emit at a wide range of 300-600 nm, which overlaps with several common fluors like BV421™, Pacific Blue, and FITC. Myeloid cell lineages tend to be particularly autofluorescent. Furthermore, stimulating cells can cause them to become more metabolically active, producing more autofluorescent proteins, vitamins, and cofactors. An unstained control sample is helpful in delineating how much autofluorescence populates your channel of interest. Also, autofluorescence doesn’t have a large Stokes shift (the difference in nanometers between the peak excitation and emission wavelengths), so tandem fluorophores can prove very useful to resolve populations on highly autofluorescent cell samples. If necessary, these factors can be taken into consideration when building a flow panel.

Stimulation Controls...

Are most appropriate when you have samples that are stimulated or exogenously treated.

Background signal can change when you stimulate samples, potentially affecting population resolution. For example, PMA stimulation can cause a decrease in surface CD4 expression in T cells while also increasing the level of autofluorescence in short wavelength emission channels with the production of autofluorescent molecules. Alternative gating strategies may have to be used (i.e., CD3+, CD8-, CD56- events) or the intracellular detection of CD4.

 

 

Human PBMCs were either untreated (left) or stimulated with 
PMA/Ionomycin for six hours (right). Samples were analyzed two days later.


In other cases, it helps to compare stimulated samples to the basal level of expression in unstimulated controls. Unstimulated controls help to establish where gates should be drawn and also let you better analyze the amount of meaningful upregulation of your target.

 

 

Human PBMCs were either untreated and stained with the full panel (orange), or stimulated with 
PMA/Ionomycin for six hours and stained with the full antibody panel (red) or an FMO for IFNγ (blue). 

 

Human Variability Controls...

Are most appropriate when you want to identify human-to-human variation.

There's a large amount of genetic variation from human to human, and this can account for some unexpected differences in expression of certain markers. This is best demonstrated in patient samples, where antigen expression, autofluorescence, and background variance often require, for the best results and accurate gating, that each fully-stained patient sample has controls derived from the same patient. Alternatively, you might consider using Veri-Cells™, a normal human control sample that can verify the consistency of reagent performance between experiments.


In the data below, two human donors display consider variability in their expression of CCR4 and CCR5. On NK cells, Donor B exhibits very low CCR4 levels, but higher levels of CCR5 when compared to donor A. In this scenario, the reagents may not be at fault. Rather, human genetic variability may explain the observed differences in staining. 

 

Live/Dead Controls...

Are most appropriate when you want to exclude dead cells and debris from your analysis.

Since dead cells and debris can non-specifically stain with antibodies and can also have antigen expression that is not consistent with live cells, it is helpful to exclude them from analysis. DNA stains like Propidium IodideHelix NP™ NIR, and Helix NP™ Green are cell membrane impermeant, meaning they only enter cells with compromised membranes, such as dead cells. For cells that will otherwise be analyzed unfixed, nucleic acid/DNA stains are the preferred live/dead indicator.

For samples that need to be fixed and permeabilized (particularly with ethanol or methanol), DNA binding dyes are often not ideal. This is because the DNA can be denatured, thereby releasing intercalated dyes and allowing them to non-specifically bind cells that are now fixed, permeabilized and thus “dead”. In addition, cells that were originally dead may lose their signal amplitude as the dyes escape. For these experiments, we highly recommend Zombie dyes, which covalently attach to primary amines instead of DNA. If the cell is alive when labeled, only amines at the cell surface will be bound. If the cells are dead when labeled, the amplitude of the signal will be several folds higher since the dye now has access to intracellular, amine-containing proteins. Zombie dyes are helpful for studies involving cytokine and transcription factor analysis requiring fixation and permeabilization.

image

One day old C57BL/6 mouse splenocytes were stained with Zombie Red™ and analyzed before fixation (purple) or after fixation and permeabilization (red). Cells alone, without Zombie Red™ staining, are indicated in black.

Veri-Cells™...

Are most appropriate when you need reference/control cells for longitudinal, multi-center studies.

Products

Reference laboratories, clinical research organizations, and multi-center clinical trials, among other institutions, need reliable controls to monitor assay performance and variability for longitudinal studies. Veri-Cells™ is our line of lyophilized control cell products, which can be used to help monitor data quality and aid in reproducibility in multi-center and longitudinal studies. 

Veri-Cells™ feature:

  • Exceptional long term stability
  • Scatter profile similar to fresh samples
  • Validation for the detection of over 150 markers

For more helpful information on Veri-Cells™, visit the FAQ page.

 

Veri-Cells™ PBMC (CD103, CD117)

Veri-Cells™ PBMC (CD103, CD117) Kit can be used as a staining control that provides assay values for CD103, CD1a, CD117, CD30, and CD71 expression. For best results, Cell Staining Buffer (Cat. No. 420201) is recommended for all washing steps. Please note that any claims relative to product performance are based on testing with BioLegend reagents and protocols. Deviations from the use of these reagents and procedures have not been assessed and may affect performance.

 

Veri-Cells™ PBMC (CD103, CD117) were stained with:

  • Fig. A: anti-human CD14 PE/Cyanine5 and anti-human CD19 FITC (right). Cells were gated on forward and side scatter (left).
  • Fig. B: anti-human CD14 PE/Cyanine5, anti-human CD19 FITC, and anti-human CD103 PE/Cyanine7 (left) or Mouse IgG1, κ PE/Cyanine7 isotype control (right). CD14- CD19- double negative single cells are shown.
  • Fig. C: anti-human CD14 PE/Cyanine5, anti-human CD19 FITC, and anti-human CD1a BV711™ (left) or Mouse IgG1, κ BV711™ isotype control (right). CD14- CD19- double negative single cells are shown.
  • Fig. D: anti-human CD14 PE/Cyanine5, anti-human CD19 FITC, and anti-human CD117 PE (left) or Mouse IgG1, κ PE isotype control (right). CD14- CD19- double negative single cells are shown. 
  • Fig. E: anti-human CD14 PE/Cyanine5, anti-human CD19 (clone HIB19) FITC, and anti-human CD30 (clone BY88) APC (left) or Mouse IgG1, κ APC isotype control (right). CD14- CD19- double negative single cells are shown.
  • Fig F: anti-human CD14 PE/Cyanine5, anti-human CD19 FITC, and anti-human CD71 BV650™ (left) or Mouse IgG2a, κ BV650™ (right). CD14- CD19- double negative single cells are shown.

Veri-Cells™ CD138 Leukocytes

Veri-Cells™ CD138 Leukocytes contain a mixture of human CD138+ cells and other leukocytes and can be used as controls to verify the expression of CD138, CD38, CD56 and CD269. In addition, Veri-Cells™ CD138 Leukocytes have been verified for expression of common markers, including CD3, CD4, CD8, CD19, CD20, CD56/16, CD14, CD25, CD69, HLA-DR, Granzyme B, Perforin, FOXP3, and Helios.

Veri-Cells™ CD138 Leukocytes were stained with anti-human CD45 (clone 2D1) PE, anti-human CD19 (clone SJ25C1) APC, anti-human CD38 (clone HIT2) FITC, and anti-human CD138 (clone MI15) Brilliant Violet 421™. CD45- and CD19- negative cells (left) were analyzed for CD138 and CD38 expression (right). Gates for the CD38 vs CD138 plot were set based on corresponding isotype controls.

 

Veri-Cells™ CD138 Leukocytes were stained with Human TBNK 6 Color Cocktail (Cat. No. 391503). CD45+ CD3+ and CD45+ CD3- cells (top right) were gated on lymphocytes (top left) and analyzed for lineage marker expression (bottom left/right).


Veri-Cells™ Heavy Metal (Ta) PBMC

Veri-Cells™ Heavy Metal (Ta) PBMCs have been labeled with the heavy metal tantalum. When mixed with sample cells, they can be separated from the cells of interest using the tantalum signal. They have been verified to stain for commonly tested surface markers including CD3, CD4, CD8, CD14, CD16, CD19, CD38, CD123, and HLA-DR.

Veri-Cells™ Heavy Metal (Ta) PBMCs were mixed with whole blood from different donors. Mixed samples were stained with a heavy-metal labeled anti-CD14 antibody and run on a CyTOF®. Median intensity of CD14 signal was measured for the blood sample alone (top row) and normalized to the median intensity of CD14 signal on Veri-Cells™ in each sample (bottom row). Normalized median values are indicated. Data provided by Adeeb Rahman, PhD, Icahn School of Medicine at Mt. Sinai.


Veri-Cells™ Activated (Cytokine) PBMC

Veri-Cells™ Activated (Cytokine) PBMC have been activated with our Cell Activation Cocktail (with Brefeldin A), which includes phorbol 12-myristate 13-acetate (PMA) and Ionomycin, for 6 hours. They have been validated to stain for activation-induced cytokines including IL-2, IL-3, IL-4, IL-5, IL-8, IL-13, IL-17, GM-CSF, IFN-γ, and TNF-α, along with surface markers typically expressed on human PBMCs. Cells do not require additional fixation nor permeabilization prior to cytokine staining, thus saving more time in your experiment.

 

Veri-Cells™ Activated (Cytokine) PBMCs were stained with anti-CD3 FITC and either anti-TNF-α PerCP/Cyanine5.5, anti-IL-17A BV421™, anti-GM-CSF Pacific Blue™, or anti-IFN-γ APC. Plots were gated based on staining with the corresponding isotype controls as indicated.


Veri-Cells™ Activated (Surface)

Veri-Cells™ Activated (Surface) have been activated with Concanavalin A for 72 hours. This makes them ideal for the detection of T cell activation markers, including CD25, CD69, CD134, CD279, CD366, and TIGIT.

Veri-Cells™ Activated (Surface) PBMCs were stained with anti-CD4 FITC and either anti-CD25 PerCP/Cyanine5.5, anti-CD134 APC, anti-CD69 APC, or anti-TIGIT APC. Plots were generated on CD3+ cells and gated based on staining with the corresponding isotype control as indicated.


Veri-Cells™ Phospho PBMC (MAPK/ERK Pathway)

Veri-Cells™ Phospho PBMC are stimulated with PMA/Ionomycin for 15 minutes. This product includes both stimulated and vehicle control cells for the staining of RAS mitogen-activated kinase pathways, including phospho-S6, phospho-p38, and phospho-ERK1/2.

 

Veri-Cells™ Phospho PBMC (MAPK/ERK Pathway) vehicle control (open histogram) or stimulated cells (closed histogram) were stained with anti-phospho S6-Alexa Fluor® 488 (left), anti-phospho p38-PE (center) or anti-human phospho ERK-Alexa Fluor® 647 (right). The histograms are gated on the lymphocyte population.


Veri-Cells™ CD34 PBMC

Veri-Cells™ CD34 PBMC contains mixture of CD34+ KG-1a cells and human PBMCs to help verify CD34 antibody binding without the need for artificial enrichment or the collection of a large number of events.

 

Veri-Cells™ CD34 PBMCs were stained with anti-CD45 (clone HI30) FITC and anti-CD34 (clone 561) PE. Dot plots have excluded doublets from analysis.


Veri-Cells™ Leukocytes 

Unlike Veri-Cells™ PBMC and CD4-Low PBMC, Veri-Cells™ Leukocytes contain neutrophils and eosinophil populations. These cells can be used as controls or reference material to monitor expression of granulocyte markers such as CD15 and CD16.

 

Veri-Cells™ Leukocytes were surface stained with anti-CD8 Alexa Fluor® 700, then fixed and permeabilized before staining with anti-Granzyme B FITC, and anti-Perforin PE. Scatter profile of the cells is shown on the left.


Veri-Cells™ CD4-Low PBMC

Veri-Cells™ CD4-Low PBMC are specially formulated to contain a lower frequency of CD4+ cells, similar to that observed in patients with CD4 immunodeficiency. The expected frequency of CD3+ CD4+ cells can be found on the certificate of analysis for that lot.

 

Veri-Cells™ PBMCs and CD4-low PBMCs were stained with anti-CD4 PE/Cyanine7 and anti-CD3 FITC and the frequency of CD4+ cells is indicated. Plots were first gated on the lymphocyte population.


Veri-Cells™ PBMC

Veri-Cells™ PBMC consist of lyophilized human PBMCs validated for over 150 surface markers and intracellular targets and exhibit a scatter profile similar to freshly isolated cells. Please refer to the Validation Tab to view the full listing of markers and clones that can be used with Veri-Cells™ PBMC.

Stability

Veri-Cells™ are highly stable, whether used immediately, 5 days after reconstitution, or up to a year in its lyophilized state, making it perfect for long-term longitudinal studies.

Consistent cell frequences pre and post-lyophilization

 

Fresh PBMCs and reconstituted Veri-Cells™ PBMC were surface stained with anti-human CD3, CD19 and CD56/CD16 antibodies. The percentage of positive cells was similar before and after the lyophilization process.

Stability after reconstitution

 

Veri-Cells™ PBMC were surface stained with anti-human CD4 PE/Cyanine7, CD8 APC, CD19 PerCP/Cyanine5.5, CD56 FITC, and CCR6 BV421™ on day 0 and day 5 post-reconstitution.

Long-term stability

 

Frequencies of CD3, CD4, CD8 positive T cells, and CD19 positive B cells were almost identical between Veri-Cells™ PBMC tested immediately post lyophilization and lyophilized Veri-Cells™ PBMC stored at room temperature for 18 months.

Excellent Surface Staining

Excellent Surface Staining

Transcription Factor Staining

Transcription Factor Staining

150+ Surface Markers Validated on Veri-Cells™ PBMC

The following cell surface markers were validated using BioLegend's LEGENDScreen™ Human PE kit.

 

 

Marker Clone
CD1c L161
CD1d 51.1
CD2 RPA-2.10
CD3 HIT3a
CD4 RPA-T4
CD5 UCHT2
CD6 BL-CD6
CD7 CD7-6B7
CD8a HIT8a
CD9 HI9a
CD11a HI111
CD11b ICRF44
CD11b CBRM1/5
CD11c 3.9
CD13 WM15
CD14 M5E2
CD16 3G8
CD18 TS1/18
CD19 HIB19
CD20 2H7
CD21 Bu32
CD22 HIB22
CD23 EBVCS-5
CD24 ML5
CD26 BA5b
CD27 O323
CD28 CD28.2
CD29 TS2/16
CD31 WM59
CD32 FUN-2
CD33 WM53
CD35 E11
CD36 5-271
CD38 HIT2
CD39 A1
CD40 HB14
CD41 HIP8
CD42b HIP1
CD43 CD43-10G7
CD44 BJ18
Marker Clone
CD45 HI30
CD45RA HI100
CD45RB MEM-55
CD45RO UCHL1
CD46 TRA-2-10
CD47 CC2C6
CD48 BJ40
CD49d 9F10
CD49e NKI-SAM-1
CD49f GoH3
CD50 CBR-IC3/1
CD52 HI186
CD53 HI29
CD54 HA58
CD55 JS11
CD56 HCD56
CD57 HCD57
CD58 TS2/9
CD59 p282 (H19)
CD61 VI-PL2
CD63 H5C6
CD64 10.1
CD69 FN50
CD73 AD2
CD74 LN2
CD79b CB3-1
CD81 5A6
CD82 ASL-24
CD84 CD84.1.21
CD85 MKT5.1
CD85d 42D1
CD85 GHI/75
CD86 IT2.2
CD87 VIM5
CD88 S5/1
CD89 A59
CD93 VIMD2
CD94 DX22
CD95 DX2
CD97 VIM3b
Marker Clone
CD99 HCD99
CD100 A8
CD101 BB27
CD102 CBR-IC2/2
CD116 4H1
CD122 TU27
CD123 6H6
CD124 G077F6
CD126 UV4
CD127 A019D5
CD132 TUGh4
CD134 Ber-ACT35 (ACT35)
CD148 A3
CD154 24-31
CD156c SHM14
CD158a/h HP-MA4
CD158b DX27
CD158e1 DX9
CD161 HP-3G10
CD162 KPL-1
CD163 GHI/61
CD164 67D2
CD165 SN2 (N6-D11)
CD172a SE5A5
CD172b B4B6
CD172g LSB2.20
CD180 MHR73-11
CD182 5E8/CXCR2
CD196 G034E3
CD197 G043H7
CD200 OX-104
CD200R OX-108
CD226 11A8
CD229 Hly-9.1.25
CD244 C1.7
CD268 11C1
CD272 MIH26
CD277 BT3.1
CD278 C398.4A
Marker Clone
CD279 EH12.2H7
CD284 HTA125
CD290 3C10C5
CD298 LNH-94
CD300e UP-H2
CD300F UP-D2
CD314 1D11
CD319 162.1
CD328 6-434
CD335 9E2
CD337 P30-15
CD352 NT-7
CD354 TREM-26
β2-microglobulin 2M2
C3aR hC3aRZ8
CLEC12A 50C1
CX3CR1 2A9-1
FcRL6 2H3
HLA-A,B,C W6/32
HLA-A2 BB7.2
HLA-DQ HLADQ1
HLA-DR L243
HLA-E 3D12
HVEM 122
IgD IA6-2
IgM MHM-88
Integrin β7 FIB504
NKp80 5D12
Siglec-9 K8
TCR gamma/delta B1
TCR Vβ13.2 H132
TCR Vβ23 αHUT7
TCR α/β IP26
TCR Vβ8 JR2 (JR.2)
TCR Vβ9 MKB1
TCR Vδ2 B6
TCR Vγ9 B3
TCR Vα7.2 3C10

100+ Surface Markers Validated on Veri-Cells™ Leukocytes

The following cell surface markers were validated using LEGENDScreen™ Human PE kit.

 

Marker Clone
β2-microglobulin 2M2
CD1c L161
CD1d 51.1
CD2 RPA-2.10
CD3 HIT3a
CD4 RPA-T4
CD5 UCHT2
CD6 BL-CD6
CD10 HI10a
CD11a HI111
CD11b ICRF44
CD13 WM15
CD14 M5E2
CD15 W6D3
CD15 (SSEA-1) MC-480
CD16 3G8
CD18 TS1/18
CD19 HIB19
CD20 2H7
CD21 Bu32
CD22 HIB22
CD23 EBVCS-5
CD24 ML5
CD26 BA5b
CD27 O323
CD28 CD28.2
CD29 TS2/16
CD31 WM59
CD32 Fun-2
CD33 WM53
Marker Clone
CD35 E11
CD38 HIT2
CD39 A1
CD40 HB14
CD43 CD43-10G7
CD44 BJ18
CD45 HI30
CD45RA HI100
CD45RB MEM-55
CD45RO UCHL1
CD47 CC2C6
CD48 BJ40
CD49d 9F10
CD49e NKI-SAM-1
CD50 CBR-IC3/1
CD52 HI186
CD53 HI29
CD55 JS11
CD56 HCD56
CD57 HCD57
CD58 TS2/9
CD59 p282 (H19)
CD64 10.1
CD66a/c/e ASL-32
CD66b G10F5
CD7 CD7-6B7
CD74 LN2
CD81 5A6
CD82 ASL-24
CD84 CD84.1.21
Marker Clone
CD85j GHI/75
CD86 IT2.2
CD87 VIM5
CD88 S5/1
CD89 A59
CD8a HIT8a
CD94 DX22
CD95 DX2
CD97 VIM3b
CD99 HCD99
CD101 BB27
CD102 CBR-IC2/2
CD116 4H1
CD122 TU27
CD127 A019D5
CD132 TUGh4
CD141 M80
CD155 SKII.4
CD156c SHM14
CD161 HP-3G10
CD163 GHI/61
CD164 67D2
CD172a/b SE5A5
CD172b B4B6
CD172g LSB2.20
CD180 MHR73-11
CD197 G043H7
CD200 OX-104
CD226 11A8
CD244 C1.7
Marker Clone
CD245 DY12
CD263 DJR3
CD268 11C1
CD278 C398.4A
CD279 (PD-1) EH12.2H7
CD284 HTA125
CD298 LNH-94
CD300e UP-H2
CD300F UP-D2
CD314 1D11
CD319 162.1
CD328 6-434
CD335 9E2
CD337 P30-15
CD354 TREM-26
CLEC12A 50C1
CX3CR1 2A9-1
DcR1 (TRAIL-R3) DJR3
Galectin-3 (Mac-2) Gal397
HLA-A,B,C W6/32
HLA-DQ HLADQ1
HLA-DR L243
IgD IA6-2
LTβR 31G4D8
NKp80 5D12
Siglec-9 K8
TCR α/β IP26
TCR gamma/delta B1
TCR Vδ2 B6
Vγ9 B3

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