The RayBiotech Cell Permeabilization Buffer is saponin-containing buffer commonly used to permeabilize cells and allow antibody to stain target intracellularly. With treatment of Brefeldin A or Monensin, inhibitors for Golgi apparatus function, cytokines will be trapped in cells and be detected by antibody in Cell Permeabilization Buffer.
RayBiotech Cell Permeabilization Buffer contains saponin and fetal bovine serum (FBS). The 10x concentrate should be diluted with dd H2O before use.
Cell Permeabilization Buffer can be stored at at 4°C in light-blocking condition, for 3 months. It can be aliquoted and kept in -20°C for long-term storage.
- Activate cells in culture in the presence of Brefeldin A (3 µg/ml) or monensin (5 µg/ml). (These are recommended concentrations. The researcher needs to titrate to determine the best concentration for their studies)
- Stain cells with live/dead markers and antibodies for surface markers.
- After surface staining, wash cells with cold PBS and fix with 4% Paraformaldehyde (PFA) for 15-30 minutes at room temperature.
- Resuspend cells in 200 µl of Cell Permeabilization Buffer, incubate the cells on ice or at 4°C for 15 minutes.
- Repeat step 4 once.
- Resuspend cells in 50 µl of Cell Permeabilization Buffer containing antibody (antibodies) against cytokines or other intracellular proteins. Incubate for 30 minutes.
- Wash cells with 250 µl of Cell Permeabilization Buffer twice and resuspend cells in FACS buffer or 1% PFA in PBS.
- Analyze by flow cytometry.
One million mouse splenocytes were activated by Ionomycin (25ng/ml) and PMA (1µg/ml) for 5 hours in culture and stained with 0.25 µg RayBright™ R647 anti-mouse CD4 and 0.5 µg RayBright™ V450 anti-mouse IFNγ in 50 µl Cell Permeabilization Buffer.
Note: RayBright™ is a trademark of RayBiotech, Inc.