Intracellular Staining With True - Phos™ Perm Buffer in Cell Suspensions
Warm Fixation Buffer ( BioLegend Cat # 420801 ) .
For each 1 x 106 cells aliquot 0.5 mL of buffer and warm to 37°C .
Chill True - Phos™ Perm Buffer to - 20°C .
For each 1 x 106 cells aliquot 1.0 ml of True - Phos™ Perm Buffer and chill to - 20°C .
Prepare a single cell suspension with the sample of interest ( Human PBMC , splenocytes , cell lines , etc ) .
Tips : 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 adding an equal volume of pre - warmed Fixation Buffer .
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 : 10 x 106 cells should be permeabilized 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 Cell Staining Buffer at a concentration of 10 x 106 cells / ml .
Transfer 100 uL ( or 1 x 106 cells ) 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 ml of Cell Staining Buffer and analyze with a flow cytometer .
