Flow Cytometry Protocol for Extracellular Membrane-Associated Proteins
Immunophenotyping suspended cells based on membrane-associated antigens is the most common use for flow cytometry. Membrane proteins are readily accessible to the antibody, therefore permeabilization steps are not required. This flow cytometry protocol for staining extracellular antigens has been developed and optimized by Bio-Techne. Individual experimental designs for flow cytometry must be optimized, including antibody dilution, incubation time and temperature (i.e. some receptors internalize with warmer temperatures). If you are concerned about target internalization, do all incubation and centrifugation steps at 4°C. For best results, use 1 x 106 cells per 100 μL of staining buffer in each sample tube. If performing surface and intracellular staining on the same sample, surface staining should be performed prior to permeabilization treatments as these may decrease surface antigen availability. Please read the protocol in its entirety before starting.
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- Fc Receptor Blocking Reagents (These include Fc receptor blocking antibodies or IgG solutions)
- Optional: Red Blood Cell Lysis Buffer (Novus Biologicals, Cat No. NBP2-29442), Human Lysis Buffer (10X) (R&D Systems, Cat No. FC-002), OR Mouse Lysis Buffer (10X) (R&D Systems, Cat No. FC-003)
- Flow Cytometry Staining Buffer (R&D Systems, Cat No. FC-001) or an equivalent solution containing BSA and sodium azide
- Primary Antibodies
- Isotype Control Antibodies
- Optional: Secondary Antibodies
- Recommended viability dye: DAPI, Novus Biologicals, Cat No. NBP2-31156; Propidium Iodide, Novus Biologicals, Cat No. NBP2-31155; or 7-AAD, Novus Biologicals, Cat No. 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
|Cells in Suspension
|After removing as much media as possible from suspended cells, add cold PBS to remove residual growth factors from cell culture media. After washing media remnants, use cells suspended in PBS and proceed with washing in Step 2.
After removing media from adherent cells, add cold PBS to remove residual growth factors from cell culture media.
- Harvest cells with a 1% BSA solution in PBS and then proceed with washing in Step 2.
- Adherent cell lines may require 0.5 mM EDTA to facilitate removal and then washed according to Step 2. Exposure time with EDTA should be minimal.
To prepare tissues for flow cytometry, mechanical and/or enzymatic disaggregation is required.
- First, mince the tissue into small sections that expose the cells and suspend in PBS. Enzymatic digestion may be required after mincing the tissue, but digestion buffer will be tissue type dependent.
- Next, pass the minced tissue suspension through a fine gauge needle several times until all cells are fully in suspension. If you experience resistance, exchange needle with a larger gauge to dissociate cells first.
Collection of whole blood into commercially available anticoagulant-treated collection tubes (EDTA or heparin) is recommended if peripheral blood cells are required for staining.
- To separate the cells from a plasma supernatant, centrifuge for 10 minutes at 1,000–2,000 x g at 4°C. Carefully remove the plasma from atop the cell pellet using a pipette. Resuspend the cell pellet in 2 mL of a 0.5% BSA in PBS wash buffer to remove plasma sample contaminants before any staining occurs (see Step 2).
- Harvest your cells (see Sample Preparation for guidance).
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
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 are 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.
- Sodium azide prevents the modulation and internalization of surface antigens. Should you opt not to use the recommended Flow Cytometry Staining Buffer (R&D Systems, Cat No. FC-001), be sure the buffer used contains sodium azide for best results of extracellular staining.
- When using whole blood samples, anti-coagulants 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.
View our complete list of flow cytometry protocols and resources.
An example of surface staining of mouse splenocytes with B220 and CD3
A) Mouse splenocytes were stained with Rat Anti-Mouse CD3 PE-conjugated Monoclonal Antibody (R&D Systems Catalog # FAB4841P) and Rat Anti-Mouse B220/CD45R APC-conjugated Monoclonal Antibody (R&D Systems Catalog # FAB1217A) or B) RatIgG2A APC-conjugated isotype control antibody (R&D Systems Catalog # IC006A).