Intracellular Staining of PUMA in Primary PBMC Lymphocytes
Dennis Juarez, David Fruman
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Abstract
Flow cytometric assessment of Intracellular PUMA levels is useful when wanting to assess PBMC subsets but are limited by sample as with primary patient samples. Here I describe a protocol for intracellular PUMA staining validated with PUMA knockdown and induction with PUMA inducing treatments.
Steps
Staining
Treat at least 1 million PBMCs per sample, including flow cytometry controls (unstained, single stain controls, and FMO controls)
Harvest cells at 20 hours and wash with PBS.
Stain cells with anti-CD3, anti-CD4 and anti-CD19 for 20 minutes at 4 degrees in the dark. Create unstained, single stain controls, and FMO controls.
After staining, wash with PBS and resuspend 1 million cells in 100uL of PBS-diluted 2% formaldehyde for 10 minutes at RT.
Add 1mL of PBS and spin down the cells at 800xg for 5 minutes.
Wash two times with 1x intracellular staining buffer (see materials).
Stain for intracellular PUMA in 1x intracellular staining buffer: 50ul staining volume per sample with 1:50 final dilution Puma (Cell signaling D30C10) Rabbit mAb
Stain at 4 degrees for 1 hour in the dark.
Spin down and wash two times with 1x intracellular buffer.
Prepare the secondary Invitrogen’s anti-Rabbit IgG 647 #A-21244 at Final Dilution 1:500 in 1x staining buffer. Be sure to stain the PUMA FMO with the secondary antibody.
Stain for 1 hr in the dark at RT.
Wash twice with 1x intracellular buffer and once with PBS. Resuspend in 100-150uL PBS and samples are ready to run.
Validation
Protocol used in PUMA knockdown cell line validated by western blot.
