Intracellular FACS staining Sample Clauses

Intracellular FACS staining. IC staining was performed using the FoxP3 staining kit (eBiosciences). Whole PBMCs were isolated and seeded at 5x105 in 48 well plates in x vivo media and 50% WPMY-1 CM. Cells were treated with 1:2 anti-CD3/28 dynabeads™ (Life technologies) for 48h and 5μg/ml brefeldin A (Sigma Xxxxxxx) was added 16h prior to harvesting. Cells were washed with FACS wash buffer and stained with CD3 and either CD4 or CD8 antibody. Cells were washed and then fixed. Cells were washed in permeabilisation buffer and the excess removed. Antibodies to the relevant cytokines were added and incubated at room temperature for 30 mins. Cells were again washed with FACS buffer and 200μls of fix buffer was added. Cells were stored at 4⁰C in the dark until acquisition on a FACS canto II (BD biosciences).
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