Extracellular Membrane Flow Cytometry Protocol

Contents

• Materials
• Sample Preparation
• Methods
• Why Use Flow Cytometry?
• Flow Cytometry Dyes
• Other Considerations

Materials

• Fc Receptor Blocking Reagents (These include Fc receptor blocking antibodies or IgG solutions)
• Optional:   Red Blood Cell Lysis Buffer (Catalog # NBP2-29442), Human Lysis Buffer (10X) (Catalog # FC-002), OR Mouse Lysis Buffer (10X) (Catalog # FC-003)
• Flow Cytometry Staining Buffer (Catalog # FC-001) or an equivalent solution containing BSA and sodium azide
• Primary Antibodies
• Isotype Control Antibodies
• Optional:   Secondary Antibodies
• Recommended viability dye: DAPI (Catalog # NBP2-31156); Propidium Iodide (Catalog # NBP2-31155); or 7-AAD (Catalog # NBP2-29446)
• 1 X PBS (0.137 M NaCl, 0.05 M NaH₂PO₄, pH 7.4)
• Bovine Serum Albumin to make 0.5% BSA in PBS as wash buffer
• Trypan Blue
• FACS Tubes (5 mL round-bottom polystyrene tubes)
• Pipettes with appropriate tips
• Centrifuge
• Vortex

Methods

1. Harvest your cells (see Sample Preparation for guidance).
2. Add 2 mL of 0.5% BSA in PBS wash buffer with a pipette to wash the cells. Centrifuge at 1,300 RPM (500 x g maximum) at room temperature (RT) for 5 minutes, decanting the supernatant. Wash a total of three times.
   • Optional: If using whole blood, a Red Blood Cell (RBC) lysis step is required at this point. This will remove RBCs allowing for identification of lymphocytes, granulocytes, and monocytes by flow cytometry. You may use RBC Lysis Buffer, Human Lysis Buffer (1X) or Mouse Lysis Buffer (1X) diluted in Flow Cytometry Staining Buffer. Add 2 mL of desired 1X Lysis Buffer, gently vortex and incubate 10 minutes at RT protected from light to lyse RBCs. Vortex intermittently. Centrifuge at 1,300 RPM (500 x g maximum) at RT for 5 minutes to pellet cells. Resuspend in 2 mL of 0.5% BSA in PBS wash buffer with a pipette to wash the cells of any remaining lysis buffer. Centrifuge at 1,300 RPM (500 x g maximum) at RT for 5 minutes to pellet cells. Discard supernatant.


3. Using a small aliquot, count the cells. Count cells using a hemocytometer and a 1:1 trypan blue exclusion stain to determine cell viability before starting.
4. Add 100 μL of Flow Cytometry Staining Buffer into FACS tubes required for your experiment. Aliquot up to 1 x 10⁶ cells per 100 μL. A separate set of cells should be prepared as a negative control alongside samples.
5. Add 1 μg blocking IgG per 1 x 10⁶ cells, gently vortex and let stand for 15 minutes at RT. Do NOT wash excess blocking IgG from this reaction.
6. Add 5-10 μL of conjugated antibody (or a previously determined amount) per 1 x 10⁶ cells and gently vortex. For unconjugated antibodies, be sure to check the data sheet for any appropriate concentrations validated for use in flow cytometry. 1 µL of primary antibody per 1 x 10⁶ cells is a good starting point. Incubate cells for 30 minutes protected from light at RT. Gently vortex intermittently to maintain a single-cell suspension.
   • Tip: If an unconjugated primary antibody was used, incubation with an appropriate secondary antibody is required. Remove any unbound conjugated antibody by washing the cells ONCE using 2 mL Flow Cytometry Staining Buffer, centrifuging at 1,300 RPM (500 x g maximum) at RT for 5 minutes to pellet cells. After washing cells to remove the primary antibody, resuspend the cell pellet in 100 μL of Flow Cytometry Staining Buffer. Add the recommended volume of secondary antibody and incubate 30 minutes protected from light. Gently vortex intermittently to maintain a single-cell suspension.


7. Remove any unbound conjugated antibody by washing cells with 2 mL Flow Cytometry Staining Buffer. Centrifuge at 1,300 RPM (500 x g maximum) for 5 minutes, decanting the supernatant.
8. Resuspend the cells in 200 – 400 μL of Flow Cytometry Staining Buffer for final flow cytometric analysis.
   • Recommended: To assess cell viability, staining using products DAPI, PI or 7-AAD should be performed after antibody staining is complete. Do NOT wash cells after this reaction. Cells must remain in the buffer during acquisition, so add dyes approximately 15 minutes prior to analysis.


9.  

Flow Cytometry Basics

Why Use Flow Cytometry?

In addition to phenotyping cells in suspension according to cellular morphology through forward and side scatter, antigen expression can be examined in various cell subtypes using flow cytometry. Forward scatter correlates with cell size, while side scatter provides details about the granularity of cells. To assess extracellular/surface antigen expression, cells can be stained by direct or indirect staining protocols. Direct staining involves a primary antibody conjugated with a fluorochrome and requires no secondary antibody for detection. Indirect staining relies on an unconjugated primary antibody forming a complex with the antigen, then a labeled secondary antibody interacts with the constant region of the primary antibody, facilitating detection. For surface proteins, it is recommended to use conjugated antibodies for direct staining. However, we have provided details on indirect staining should your experiment require these steps.

Flow Cytometry Dyes

When selecting your fluorochrome conjugates, you should select the dyes based on the predicted expression of the antigen. To help with your conjugate selection, this subset of fluorochromes is listed from the brightest to lowest intensity: PE, PE-Cy7, PE-Cy5, APC, APC-Cy7, Alexa Fluor^® 647, Alexa Fluor^® 700, FITC, Alexa Fluor^® 488. For highly expressed antigens, conjugates with lower intensities are recommended. Use the brightest conjugate for the lowest expressing antigen in your panel. To add multiple fluorescent probes and determine compatibility, use the Flow Cytometry Spectra Viewer. Our Flow Cytometry Panel Builder can help determine which conjugated antibodies may work best for your experimental design. Tandem dyes are only recommended for extracellular staining given their size and inability to efficiently cross the plasma membrane for intracellular staining. It is critical to use isotype controls, Fluorescence-minus-one (FMO) controls or single-color controls for compensation when gating, particularly when tandem dyes are used in flow cytometry. Learn more about Tandem Dyes here.

Other Considerations

• Sodium azide prevents the modulation and internalization of surface antigens. Should you opt not to use the recommended Flow Cytometry Staining Buffer (Catalog # FC-001), be sure the buffer used contains sodium azide for best results of extracellular staining.
• When using whole blood samples, anticoagulants are required. Heparin can stimulate white blood cells to release cytokines. If cytokines will be measured in your intracellular analysis consider using EDTA coated collection tubes.
• In general, centrifugation speeds that result in cells becoming difficult to resuspend should be avoided. We recommend using 1,300 RPM but no greater than 500 x g based on your centrifuge settings.
• Viability dyes are highly recommended, particularly if cell suspensions are from processed tissue. Tissue dissociation and digestion often results in cell death, thus it is important to discern between viable and dead cells during analysis. If cells will be fixed and/or permeabilized, viability dyes should be used before cell fixation.