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
