7 Tips for Optimizing Your COVID-19 Flow Cytometry Experiments

COVID-19 infections trigger a myriad of immune responses involving several types of immune cells. Flow cytometry is a powerful technique for studying COVID-19 immune responses as it provides the ability to measure multiple proteins simultaneously at the single-cell level. Flow cytometry can be used to assess immunophenotypic and functional changes (cytokine production, proliferation, cytotoxicity) in response to viral infection.

Here are some excerpts from the COVID-19 ebook that our scientists put together for accelerating your COVID-19 research. We provide several tips and resources for avoiding pitfalls and optimizing your flow cytometry experiments.

If you are interested in learning more about our predesigned multicolor panels for the identification of various immune cell subtypes, such as naïve and memory T cell subsets, senescent and exhausted T cell subsets, regulatory T cells, B cells, monocytes, and more, you can download our COVID-19 ebook.

1. Selecting your sample type and picking the right lysis solution

The sample type used for a given experiment may vary depending on the cell population of interest and/or the biological application. Here are some general guidelines for choosing the right sample type for your application.

Some buffers for RBC lysis (BD FACS™ Lysing Solution) contain a fixative buffer, whereas others (BD Pharm Lyse™ Lysing Buffer) do not. The potential impact on antigen integrity and compatibility with viability stains need to be taken into consideration when using RBC lysis solutions containing a fixative buffer. BD Pharm Lyse™ Lysing Buffer and BD FACS™ Lysing Solution work optimally when EDTA is used as an anticoagulant instead of heparin. 
 

• Keep cells on ice after RBC lysis with BD FACS™ Lysing Solution to preserve granulocyte morphology.
• Perform RBC lysis with BD FACS™ Lysing Solution post antibody staining if antigens of interest may be impacted by a fixative.
• Use the BD Vacutainer^® CPT™ Mononuclear Cell Preparation Tube to facilitate the PBMC isolation workflow.

2. Antibody test size and titration

To save you time and cell samples, pretitrated test size reagents are bottled at an optimal concentration, with the best signal-to-noise ratio on relevant models. Technical data sheets provide data generated on the relevant primary model at this optimal concentration.

• For antibodies sold by mass (e.g., 0.2 mg/mL), antibody titration is required to determine the optimal concentration for a given cell type and application.
• Consult the technical data sheet of your test size antibody to know the sample type used to predetermine the optimal concentration
• If you are using a different sample type or experimental conditions, you may need to perform your own titration to determine the optimal concentration for your specific application
  
  

3. Antibody staining

The protocols, reagents and workflow required for an optimal antibody staining depend on the cellular localization of the antigen of interest, i.e., surface or intracellular markers, as well as the impact of buffers on antigen and fluorochrome integrity.

• Use BD Horizon™ Brilliant Stain Buffer for optimal staining when using fluorescent dyes such as BD Horizon Brilliant™ Blue, BD Horizon Brilliant™ UV and/or BD Horizon Brilliant™ Violet Dyes.
• Use of BD Horizon™ Brilliant Stain Buffer Plus is recommended to reduce test volume for applications where total staining volume is a concern.
• Resolution of chemokine receptors may be improved by staining cells at 37 °C for 10 minutes. Add the antibody cocktail for the remaining surface markers and continue with your staining protocol.
• Be aware of potential adverse effects of different fixation and permeabilization buffers on surface antigens and fluorochromes.

4. Cell activation

Immune cell activation is often required to induce cytokine production and detection via flow cytometry. The protocols and reagents for cell activation vary depending on the cytokine and species of interest. 
 

• Be aware of potential downregulation/internalization of some surface markers (i.e., CD3, CD4) when using stimuli such as phorbol 12-myristate 13-acetate (PMA).
• Titrate the concentration of stimulus and incubation time to ensure resolution of potentially impacted surface markers and sufficient cytokine production.
• Use bright fluorochromes and/or perform intracellular staining for downregulated and/or internalized markers.

5. Intracellular cytokine staining

Protein transport inhibitors may be required to trap cytokines inside the cells prior to intracellular cytokine analysis. The choice between two common protein transport inhibitors, monensin (BD GolgiStop™) and brefeldin A (BD GolgiPlug™), as well as the incubation time, depend on the cytokine and species of interest.
 

• Let cells incubate or culture with the stimulus for at least an hour before adding the protein transport inhibitor.
• Be aware of cell toxicity upon prolonged exposure to protein transport inhibitors (>18 hours).
• If prolonged incubation time is required, use the less toxic BD GolgiPlug™ Inhibitor.
• When investigating multiple cytokines, you can incubate the cells with both BD GolgiStop™ and BD GolgiPlug™ Inhibitors for optimal resolution of each cytokine.
  
  

6. Exclusion of dead cells using viability stains

Viability stains enable exclusion of dead cells that could introduce staining artifacts or alter protein expression patterns. This is particularly important when analyzing samples with high amounts of dead cells, such as activated or tissue-derived cells. The choice between two main categories of viability stains, nucleic acid stains and fixable viability stains (FVS), depends on the experimental workflow.
 

• Perform stain with FVS before fixation in protein-free buffer (e.g., 1X PBS) to avoid dye sequestration and suboptimal staining.
• Wash FVS-stained cells with protein containing buffer (e.g., 1X PBS with FBS or BSA) to eliminate unbound dye and reduce background.
• FVS titration is recommended for specific cell types and/or applications.
  
  

7. Absolute cell count and antigen quantification

Flow cytometry analysis conventionally provides information about the relative frequency of populations of interest and intensity of fluorescent signal (median fluorescence intensity, MFI). The BD Trucount™ Absolute Counting Tubes and BD Quantibrite™ PE Phycoerythrin Fluorescence Quantitation Kit are companion products developed specifically to enable absolute cell count and surface antigen quantification, respectively.
 

• When using BD Trucount™ Tubes, use a buffer with protein in it (e.g., 1X PBS with FBS or BSA) to avoid cell clumping and inaccurate counts.
• Stain whole blood by following a lyse/no-wash procedure when using BD Trucount™ Tubes to avoid potential cell loss and inaccurate counts.
• Antigen quantification using the BD Quantibrite™ PE Phycoerythrin Fluorescence Quantitation Kit is based on the assumption of a fluorochrome-to-antibody ratio of 1:1.

If you are interested in learning more about predesigned multicolor panels for the identification of various immune cell subtypes, download our COVID-19 ebook.