Tissue samples were collected and transported in RPMI-1640 (Sigma, cat# R0883-500ML). Single cell suspensions were produced by enzymatic digestion using liberase with subsequent cellular disaggregation using a Miltenyi gentleMACS Octo Dissociator. Lymphocytes were isolated from single cell suspension by gradient centrifugation on Ficoll Paque Plus (GE Healthcare, cat# 17-1440-03) and stored in liquid nitrogen. Blood samples were collected in BD Vacutainer EDTA blood collection tubes (BD cat# 367525), PBMC’s were then isolated by gradient centrifugation on Ficoll Paque (GE Healthcare, cat# 17-1440-03) and stored in liquid nitrogen.
FC receptors were blocked with Human Fc Receptor Binding Inhibitor (Thermo) before staining. Non-viable cells were stained using the eBioscience Fixable Viability Dye eFluor 780 (Thermo). Cells were stained in BD Brilliant stain buffer (BD cat# 563794) with the following monoclonal antibodies: anti-human CD3 (clone SK7, BD cat# 565511), anti-human CD4 (clone SK3, BD cat# 566003), anti-human CD8 (clone RPA-T8, BD cat# 564804). Data was acquired on a BD Symphony flow cytometer and analyzed in FlowJo. Cells were gated for size, single cells, live cells, CD3+CD8+ T cells.
FC receptors were blocked with Human Fc Receptor Binding Inhibitor (Thermo) before staining. Non-viable cells were stained using the eBioscience Fixable Viability Dye eFluor 780 (Thermo). Cells were stained in BD Brilliant stain buffer (BD cat# 563794) with the following monoclonal antibodies: anti-human CD3 (clone SK7, BD cat# 565511), anti-human CD4 (clone SK3, BD cat# 566003), anti-human CD8 (clone RPA-T8, BD cat# 564804). Data was acquired on a BD Symphony flow cytometer and analyzed in FlowJo. Cells were gated for size, single cells, live cells, CD3+CD8+ T cells.