Activation and Intracellular Staining of Whole Blood : For the Detection of Intracellular Cytokines and Other Intracellular Targets
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 cells in Intracellular Staining Perm Wash Buffer and centrifuge at 350 x g for 5 - 10 minutes .
( 2 / 3 ) Resuspend fixed cells in Intracellular Staining Perm Wash Buffer and centrifuge at 350 x g for 5 - 10 minutes .
( 3 / 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 .
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 ) 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 .
Resuspend fixed and intracellularly labeled cells in 0.5 ml Cell Staining Buffer and analyze with appropriate controls .
Set PMT voltage and compensation using cell surface staining controls .
Set quadrant markers based on blocking controls , isotype controls , or unstained cells .
Dilute heparinized whole blood 1 : 1 with sterile appropriate tissue culture medium .
At this stage , in vitro cellular stimulation by either antigen or mitogen can be performed .
If intending to stain intracellular cytokines or chemokines ( e . g . IFN - γ or IL - 4 ) , addition of an efficient protein transport inhibitor such as brefeldin A ( BioLegend Cat . No . 420601 ) or monensin ( BioLegend Cat . No . 420701 ) is critical .
After addition of a suitable cellular activator , aliquot 200 μl of the whole blood cell suspension into 12 x 75 mm plastic tubes and incubate for 4 - 6 hours in 5 % CO2 at 37°C .
Add 2 ml of 1X Red Blood Cell Lysis Buffer ( Cat . No . 420301 ) and incubate for 5 - 10 minutes at room temperature .
Centrifuge at 350 x g for 5 minutes and discard the supernatant .
Wash cells 1X with Cell Staining Buffer .
