Anti cd8 apc a700

Peripheral blood mononuclear cells (PBMCs) were separated by density centrifugation and cryopreserved. Prior to analysis on a Beckman Coulter Gallios flow cytometer with seven color acquisition, cryopreserved PBMC samples were thawed and cultured for 24 h before use to allow cells to rest and re-express cell surface molecules. Cells were stained with Aqua Live/Dead cell exclusion dye (Invitrogen) and the following fluorochrome-conjugated antibodies: anti-CD3-allophycocyanin (APC) (Becton Dickinson), anti-CD4-V450 (Becton Dickinson), anti-CD8-APC-A700 (Beckman Coulter), anti-CD45RA-phycoerythrin-Cy7 (Becton Dickinson), anti-CD62L-APC-Cy7 (BioLegend) and anti-CCR7-peridinin chlorophyll protein-Cy5.5 (Becton Dickinson). Single labeled tubes for each antibody, isotype-matched control antibodies, fluorescence-minus-one controls, dead cell exclusion and doublet discrimination were used to ensure accurate positive cut-off values and compensation matrices and to validate cell phenotype detection sensitivity and resolution. Figure 1 shows the gating strategy used to identify the memory T cell subsets. Flow cytometry data was analyzed using Kaluza 1.2 software (Beckman Coulter).