1. Purpose
This standard operating procedure (SOP) outlines the procedure for the preparation and staining of cells for flow cytometry analysis. The goal is to standardize the flow cytometry staining process, ensuring accurate and reproducible results.
2. Scope
This procedure is applicable to the staining of suspension cells for flow cytometry analysis. It is intended for use with cells cultured in standard media and a variety of fluorochrome-conjugated antibodies for surface or intracelluar marker detection.
3. Materials and equipment
Cells
PBS
FACS buffer (PBS with 2% FBS)
Antibodies (fluorochrome-conjugated)
Centrifuge
Vortex mixer
Ice (to maintain cell temperature during incubation)
Flow cytometer
Pipettes and tips
4. Procedure
4.1 Harvesting cells
For suspension cells, centrifuge at 300-400 x g for 5-10 minutes at 4°C to pellet the cells.
Discard the supernatant carefully, and resuspend the cell pellet in FACS buffer (1-2 x 106 cells/mL).
Spin again, discard the supernatant, and re-suspend the cells in FACS buffer (e.g., 250 µL/wash for micro-well plates, 1 mL/wash for tubes).
4.2 Blocking Fc receptors (Optional)
- If needed, block non-specific binding by incubating cells with Fc receptor blocker (e.g., human or mouse IgG) at 4°C for 10-20 minutes.
4.3 Staining cell surface antigens
Prepare the antibody cocktail in FACS buffer.
Add the antibody cocktail to the re-suspended cells.
Incubate the cells with an antibody mixture for 30 minutes at 4°C in the dark.
Gently vortex the sample every 10-15 minutes to ensure even antibody distribution.
After antibody incubation, wash the cells by centrifuging at 300-400 x g for 5-10 minutes at 4°C.
Discard the supernatant and resuspend the pellet in FACS buffer.
Repeat the washing step 1-2 times to remove excess antibody.
4.4 Fixing and permeabilizing cells (Optional)
This step is optional and is typically performed when intracellular staining is required (e.g., detecting intracellular proteins, transcription factors, etc.).
Thoroughly resuspend cells and add 100 µL per well for microwell plates (or 250 µL for tubes) of Fixation/ Permeabilization solution for 20 minutes at 4°C.
Wash cells two times in 1× BD Perm/Wash™ buffer (e.g., 1 mL/ wash for staining in tubes and 250 µL/wash final volume for staining in microwell plates) and pellet.
4.5 Staining for intracellular cytokines
Thoroughly resuspend fixed/permeabilized cells in 50 µL of BD Perm/Wash™ buffer containing a pre-determined optimal concentration of a fluorochrome-conjugated anti-cytokine antibody or appropriate negative control. Incubate at 4°C for 30 minutes in the dark.
Wash cells 2 times with 1× BD Perm/Wash™ buffer (1 mL/wash for staining in tubes and 250 µL/wash final volume for staining in microwell plates) and resuspend in Staining Buffer prior to flow cytometric analysis.