Using PBMC as a Quality Control for Flow Cytometry

Our cryopreserved peripheral blood mononuclear cells are a great tool for flow cytometry assays. Whether you want to confirm your antibodies are working properly or measure for consistency, our PBMC are the tools you need for quality control. 

Using PBMC to Test Antibodies

Sometimes you might not be sure if your chosen reagent is correctly staining the targeted antigen. To confirm your reagent is working, use PBMC as a control. 

If the stained PBMC looks as expected, you can be confident that the antibody reagent you are using is working. Since we include data from our quality control testing, you can use those results as a guide in your own lab.

Example 1: Staining of CD8

If you wanted to confirm appropriate staining of CD8, you can take advantage of the fact that it is expressed on T cells but is not expressed by CD4+ T cells. That means that if you perform 3 color staining with antibodies to CD3, CD4, and an antibody to CD8, you should find that all of the CD8+ cells are also CD3+ and that the CD4+ cells are negative for CD8.

Example 2: Staining of PD-1

Staining of PD-1 is often rather dim and it can be difficult to convince yourself that the staining you are observing is “real.”  At least that’s how we felt about it. We set up an experiment in which we performed staining for PD-1 and used anti-CD3 to focus on the T cells. We then stimulated the T cells with PHA and followed staining of PD-1 over the course of 4 days of culture. See the graph below for our results.

Figure 1. Staining of PD-1 on CD3+ T cells following stimulation with PHA, a T cell mitogen. The filled histogram shows the level of staining in the PBMC prior to stimulation. The red line is after 24 hours, blue is 48 hours, purple is 72 hours, and green is 96 hours. This shows that PD-1 expression is increased after 48 hours and then declines over the next 2 days.

Flow Cytometry Tips

  • Our PBMC can be used as a negative or positive control to your target cell population which is verified by our Certificate of Analysis.
  • Reduce day-to-day variation of MFI of antibody targets by using our PBMC as a reference standard without wasting your precious samples.
  • Ensure that you have proper controls which may include IgG, negative or random antibody epitope, or Fluorescence Minus One (FMO) controls which can give you confidence that you have identified positive target population. 

Using PBMC to Measure Flow Cytometry Consistency

Before running your assay, be sure your flow cytometer and methods are producing consistent results. Our large lots of PBMC make it easy to procure dozens of vials of the same product for these types of quality control experiments.

Versatility and Ease of Use in Calibration of Immunophenotyping by Using Astarte PBMC

Figure 2. A wide range of cell types occur in our PBMC. A) Leukocyte marker CD45 and T cell marker CD3 show distinct populations along with NK marker CD56 in our PBMC. B) If we subdivide the T cells, we can quantitate CD4 and CD8 T cell populations. C) A hot research topic currently is checkpoint markers, by using gates of CD4 helper T cells and CD8 cytotoxic T cells from previous gate (B) we can identify TIGIT positive T cells are also mostly CD8+ in donor 455. Using the histogram to compare IgG control (red) to TIGIT stained population (green) shows bimodal population when compared to IgG1 control. D) PBMC represent versatility in calibration of FACS data by use of such a broad range of cell types, even relatively small populations, such as Gamma-Delta T cells. (Acquired on FACS Canto, Lot #4546JA20)

Run one vial of PBMC through the flow cytometer to confirm the proper staining of the marker of interest. Because each vial contains the same cells, the staining of the control cells can assure you that the instrument settings are appropriate. Run these control tests frequently to be sure you are seeing consistent results before resuming your assay. 

Flow Cytometry Tips

  • Control fluorescence spillover using the auto compensation feature. Alternatively, try to export your FCS files in version 3.0 or later. These versions allow for post-acquisition compensation using 3rd-party programs, such as FLOWJo or FCSexpress, or the free Chromocyte FCSanalyzer. Auto Compensation is a far more common accepted practice that removes human variation and is considered superior to the traditional “By Eye” compensation. 
  • If possible, titrate your antibodies to reduce the lot-to-lot variation and reduce consumption. It helps to use the same lot of PBMC, a low-cost solution for standardization, to validate variation in staining intensity experienced between lots of antibodies.  
  • Determine whether there is cross reactivity between the antibodies you intend to use prior to starting a new assay, and use a block if necessary.

Let Us Develop Your Next Assay

Give us a desired output and we will craft a custom assay to help you understand the functionality or effect of your compound or drug. 

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