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.
