Cd8 buv805

Thawed cells were resuspended in 2% FBS/PBS and plated in a 96-well V-bottom plate. Antibody cocktails were prepared in FACS buffer (0.1% BSA/0.1% sodium azide/PBS). Cells were pelleted by centrifugation at 490 g for 5 min at 4 °C. Cells were then stained with 50 μL of Zombie UV Fixable viability dye (diluted 1/500 in PBS) for 20 min on ice in the dark. Cells were washed 3 times with FACS buffer. Cells were then incubated with 50 μL of blocking agents (normal mouse serum 1/20, Fc block 1/10) for 15 min on ice. Antibody cocktails were prepared in FACS Buffer and 50 μL added to each sample and then incubated for 30 min on ice in the dark. Cells were washed 3 times in FACS buffer and then fixed by resuspending in 150 μL of 1% formaldehyde for 20 min at room temperature. Cells were then washed and resuspended in FACS buffer and run on FACSymphony (BD Pharmingen). Samples were analyzed using FlowJo software (Tree Star).
Intracellular staining for MX-1 was performed for 1 × 10⁵ thawed PBMC using the Transcription Factor Buffer Set (BD Biosciences) according to the manufacturer's directions. Permeabilized cells were stained with CD3-PerCP-Cy5.5, CD4-BUV395, CD8-BUV805, CD45RA-BUV737, CD27-APC-R700 (BD Biosciences) and 1 μg MX-1-AF647 (Abcam) according to manufacturer's directions and analyzed on a 5-laser Fortessa X20 (BD Biosciences) as previously described [30 (link)].