Intracellular Staining With True-Phos™ Perm Buffer in Cell Suspensions Protocol
Protocol Steps
Buffer Preparation:
- Warm Fixation Buffer (BioLegend Cat 420801). For each 1 x 106 cells aliquot 0.5mL of buffer and warm to 37°C.
- Chill True-Phos™ Perm Buffer to -20°C. For each 1 x 106 cells aliquot 1.0ml of True-Phos™ Perm Buffer and chill to -20°C.
Sample Preparation:
- 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 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.
- 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.
- While vortexing, permeabilize cells by adding pre-chilled True-Phos™ Perm Buffer. Example: 10 x 106 cells should be permeabilized with 10mL of pre-chilled True-Phos™ Perm Buffer.
- 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.
- Centrifuge cells at 1000xg 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 1000xg 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 100µL (or 1 x 106 cells) to a 12 x 75mm tube.
- Add antibody cocktail(s) to appropriate tubes, vortex to mix, and incubate for 30 minutes at room temperature in the dark.
- 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.
- Resuspend cells in approximately 500µl of Cell Staining Buffer and analyze with a flow cytometer.