Intracellular Staining With True-Phos™ Perm Buffer in Whole Blood
Warm 1 X RBC Lysis/Fixation Solution (Cat# 422401, 10X solution).
For each 0.1 mL of whole blood, aliquot 2 mL of 1 X RBC Lysis/Fixation Solution to a 50 mL conical tube and warm to 37°C.
Chill True-Phos™ Perm Buffer to -20°C.
For each 0.1 mL of whole blood, aliquot 1.0 mL of True-Phos™ Perm Buffer and chill to -20°C.
Aliquot 0.1 mL of whole blood (heparin) into a 50 mL conical tube for each test Tips: - 22 tests (or 2.2 mL of whole blood) are the maximum number of tests that can be processed in a 50 mL conical, due to volume constraints.
-Prepare two aliquots: Negative control: untreated, Positive control: treated with stimuli 
-Incubate the cells with the appropriate stimuli, at the suitable temperature and time.
Fix the cells immediately after treatment by pre-warmed 1 X RBC Lysis/Fixation Solution.
Gently pipette to ensure thorough mixing. 
Incubate at 37°C for 15 minutes to ensure cells are properly fixed. 
Centrifuge cells at 350 x g at room temperature for 5 minutes, decant supernatant, vortex to resuspend cell pellet. 
Add sufficient Cell Staining Buffer to wash the cells (approximately 2 ml for each 1 x 106 cells, BioLegend Cell Staining Buffer recommended, Cat#420201), centrifuge at 350 x g at room temperature for 5 minutes, and decant supernatant.
Repeat, for a total of two washes. 
Gently pipette cells using residual volume to resuspend cell pellet. 
While vortexing, permeabilize cells by adding pre-chilled True-Phos™ Perm Buffer. 
Example: for 1 mL of whole blood, permeabilize with 10 mL of pre-chilled True-Phos™ Perm Buffer. 
Incubate at -20°C for 60 minutes to ensure cells are properly permeabilized. 
Centrifuge cells at 1000 x g at room temperature for 5 minutes, decant supernatant, vortex to resuspend cell pellet. 
Add sufficient Cell Staining Buffer to wash the cells, centrifuge cells at 1000 x g at room temperature for 5 minutes, decant supernatant.
Repeat, for a total of two washes. 
Resuspend the cells in a volume of Cell Staining Buffer equivalent to the starting volume of blood. 
Example: if starting volume of whole blood was 1 mL, resuspend cell pellet in 1 mL of Cell Staining Buffer. 
Transfer 100 ml to a 12 x 75 mm tube. 
Add antibody cocktail(s) to appropriate tubes, vortex to mix, and incubate for 30 minutes at room temperature in the dark. 
Add 2 mL of Cell Staining Buffer, centrifuge cells at 1000 x g at room temperature for 5 minutes, decant supernatant.
Repeat, for a total of two washes. 
Resuspend cells in approximately 500 uL of Cell Staining Buffer and analyze with a flow cytometer
