The RayBio^® Fixation/Permeabilization Buffer contains paraformaldehyde, saponin and FBS, commonly used to fix and permeabilize cells simultaneously and allow further antibody staining intracellularly.

1. Activate cells in culture in the presence of Brefeldin A (1:1000 dilution in media) or monensin (1:1000 dilution in media).
2. Wash the cells with PBS, spin down the cells at 500g, and then stain cells with live/dead markers and antibodies for surface markers.
3. After surface staining, wash cells with cold PBS and incubate with RayBio Fixation/Permeabilization Buffer (Catalog #: 137-00015) for 15-30 minutes at room temperature.
4. Spin down the cells at 500g, wash the cells with 200 µl of Cell Permeabilization Buffer (Catalog #: 137-00006).
5. Repeat step 4 once.
6. Spin down the cells at 500g, resuspend cells in 50 µl of Cell Permeabilization Buffer containing antibody (antibodies) against cytokines or other intracellular proteins. Incubate for 30 minutes.
7. Wash cells with 250 µl of Cell Permeabilization Buffer twice and resuspend cells in FACS buffer or 1% PFA in PBS.
8. Analyze by flow cytometry.

Stored at 2 °C - 8 °C, protected from prolonged light exposure. DO NOT FREEZE.

Note: RayBright® is a trademark of RayBiotech, Inc.