TITLE: Intracellular Staining With True-Phos™ Perm Buffer in Whole Blood
AUTHORS: Sam Li

[DESCRIPTION]
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[STEPS]
?. [Buffer Preparation:]
Warm 1 X RBC Lysis/Fixation Solution (Cat 422401, 10X solution). For each 0.1mL of whole blood, aliquot 2mL of 1 X RBC Lysis/Fixation Solution to a 50mL conical tube and warm to 37°C.
?. [Buffer Preparation:]
Chill True-Phos™ Perm Buffer to -20°C. For each 0.1mL of whole blood, aliquot 1.0mL of True-Phos™ Perm Buffer and chill to -20°C.
?. [Sample Preparation:]
Aliquot 0.1mL of whole blood (heparin) into a 50mL conical tube for each test.Tips:22 tests (or 2.2mL of whole blood) are the maximum number of tests that can be processed in a 50mL 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.
?. [Sample Preparation:]
Fix the cells immediately after treatment by pre-warmed 1 X RBC Lysis/Fixation Solution. Gently pipette to ensure thorough mixing.
?. [Sample Preparation:]
Incubate at 37°C for 15 minutes to ensure cells are properly fixed.
?. [Sample Preparation:]
Centrifuge cells at 350xg at room temperature for 5 minutes, decant supernatant, vortex to resuspend cell pellet.
?. [Staining with Specific Antibodies:]
Add sufficient Cell Staining Buffer to wash the cells (approximately 2ml for each 1 x 106 cells, BioLegend Cell Staining Buffer recommended, Cat 420201), centrifuge at 350xg at room temperature for 5 minutes and decant supernatant. Repeat, for a total of two washes.
?. [Staining with Specific Antibodies:]
Gently pipette cells using residual volume to resuspend cell pellet. Note: if cells are not fully resuspended, True-Phos™ Perm Buffer addition will cause significant cell loss.
?. [Staining with Specific Antibodies:]
While vortexing, permeabilize cells by adding pre-chilled True-Phos™ Perm Buffer.Example: for 1mL of whole blood, permeabilize with 10mL of pre-chilled True-Phos™ Perm Buffer.
?. [Staining with Specific Antibodies:]
Incubate at -20°C for at least 60 minutes to ensure cells are properly permeabilized. Note: cells can be stored in the True-Phos Perm Buffer overnight at -20°C.
?. [Staining with Specific Antibodies:]
Centrifuge cells at 1000xg at room temperature for 5 minutes, decant supernatant, vortex to resuspend cell pellet.
?. [Staining with Specific Antibodies:]
Add sufficient Cell Staining Buffer to wash the cells, centrifuge cells at 1000xg at room temperature for 5 minutes, decant supernatant. Repeat, for a total of two washes.
?. [Staining with Specific Antibodies:]
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.
?. [Staining with Specific Antibodies:]
Transfer 100µL to a 12 x 75mm tube.
?. [Staining with Specific Antibodies:]
Add antibody cocktail(s) to appropriate tubes, vortex to mix, and incubate for 30 minutes at room temperature in the dark.
?. [Staining with Specific Antibodies:]
Add 2mL of Cell Staining Buffer, centrifuge cells at 1000xg at room temperature for 5 minutes, decant supernatant. Repeat, for a total of two washes.
?. [Staining with Specific Antibodies:]
Resuspend cells in approximately 500µl of Cell Staining Buffer and analyze with a flow cytometer.