Intracellular Flow Cytometry Staining Protocol: For the Detection of Intracellular Cytokines and Other Intracellular Targets
Resuspend fixed cells in Intracellular Staining Perm Wash Buffer and centrifuge at 350 x g for 5-10 minutes.
(2/3) If staining intracellular antigens (e.g. IFN-γ or IL-4), first perform cell surface antigen staining as described in BioLegend’s Cell Surface Immunofluorescence Staining Protocol, then fix cells in 0.5 ml/tube Fixation Buffer (BioLegend Cat. No. 420801) in the dark for 20 minutes at room temperature.
Centrifuge at 350 x g for 5 minutes, discard supernatant.
To put the experiment “on hold” at this point for future staining and analysis, wash cells 1x with Cell StainingBuffer (BioLegend Cat. No. 420201).
Resuspend cells in Cell Staining Buffer and store cells at 4°C (short term) or in 90% FCS/10% DMSO for storage at -80°C (long term, for fixed cells without surface antigen staining).
Dilute 10X Intracellular Staining Perm Wash Buffer (Cat. No. 421002) to 1X in DI water.
Resuspend fixed cells in Intracellular Staining Perm Wash Buffer and centrifuge at 350 x g for 5-10 minutes.
(1/3) Resuspend fixed/permeabilized cells in residual Intracellular Staining Perm Wash Buffer and add a predetermined optimum concentration of fluorophore-conjugated antibody of interest (e.g. PE anti-IFN-γ) or an appropriate negative control for 20 minutes in the dark at room temperature.
If primary intracellular antibody is biotinylated, it will be necessary to perform fluorophore conjugated Streptavidin incubations and subsequent washes in Intracellular Staining Perm Wash Buffer.
Wash with 2 ml of Intracellular Staining Perm Wash Buffer and centrifuge at 350 x g for 5 minutes.
(1/2) Wash with 2 ml of Intracellular Staining Perm Wash Buffer and centrifuge at 350 x g for 5 minutes.
(2/2) Resuspend fixed and intracellularly labeled cells in 0.5 ml Cell Staining Buffer and analyze with appropriate controls.
Resuspend fixed cells in Intracellular Staining Perm Wash Buffer and centrifuge at 350 x g for 5-10 minutes.
(3/3) Set PMT voltage and compensation using cell surface staining controls.
Set quadrant markers based on blocking controls, isotype controls, or unstained cells.
