Totalseq a0112

For isolation of CD8 T cells 10 days after infection, single-cell suspensions were generated from four mice per recipient group by macerating spleens through nylon filters. CD8 T cells were enriched from these suspensions using a Stemcell EasySep™ Mouse CD8 T Cell Isolation Kit (#19853). These samples were stained to block Fc receptors then stained with antibodies and live/dead stain (LIVE/DEAD™ Fixable Violet Dead Cell Stain Kit, ThermoFisher # L34955) for 30 minutes on ice shielded from light. The antibodies used for cell surface staining from BioLegend were as follows; PE anti-mouse CD8β Antibody (YTS156.7.7), APC anti-mouse CD45.1 Antibody (A20) and APC anti-rat CD90/mouse CD90.1 (Thy-1.1) Antibody (OX-7). Samples were subsequently washed twice and ~1X10⁶ congenically marked OT-I cells were purified using fluorescence activated cell sorting for each group of recipients. The samples purified in this way from each group of recipients were then suspended in 100μL buffer and labeled with 1μg per sample of the following Total-seq A antibodies from BioLegend: TotalSeq™-A0198 anti-mouse CD127 (A7R34), TotalSeq™-A0250 anti-mouse/human KLRG1 (2F1/KLRG1), TotalSeq™-A0073 anti-mouse/human CD44 (IM7) and TotalSeq™-A0112 anti-mouse CD62L (MEL-14). Samples were labeled for 30 minutes on ice and subsequently washed with 1mL PBS twice.