Anti mouse cd8α pe cy7

HI rWR (A3 or F17 where indicated) or UV-attenuated WR-mini-OVA was used to infect either MutuDCs, 293A cells, or 293KbC2 cells (57 (link)) for 2 h with rocking at an MOI of 10 before the inoculum was replaced with IMDM-10. Splenocytes from a naive OT-I mouse were harvested and subjected to CD8α-negative enrichment (Miltenyi Biotech, 130-096-543). The eEnrichment efficiency was checked by flow cytometry, and samples were at least 85% CD8α⁺ Vα2⁺ T cells. OT-I CD8⁺ T cells were added to V-bottom plates in D10 with β-mercaptoethanol, and infected cells were added in the same medium at a ratio of 1:2 (infected cell target to OT-I). After 24 h, the cells were labeled with anti-mouse CD11c-FITC (BioLegend, clone N418), anti-mouse CD8α-PE-Cy7 (BioLegend, clone 53.67), anti-mouse TCR Vα2-APC (BioLegend, clone B20.1), anti-mouse CD69-PerCP-Cy5.5 (BioLegend, clone H1.2F3), and anti-mouse CD25 (BioLegend, clone 3C7) antibodies diluted in PBS with 2% FBS. Events were gated sequentially on SSC-A × FSC-A (lymphocytes), FSC-H × FSC-W (single cells), SSC-H x SSC-W (single cells), SSC-A × FITC/GFP^– (MutuDC exclusion), PE-Cy7 × APC (CD8⁺ Vα2⁺ cells), and PE × PerCP-Cy5.5 (CD25⁺ CD69⁺ activated cells) to determine the percentages of CD8⁺ Vα2⁺ T cells that upregulate activation markers.

To isolate OT-1 transgenic CD3⁺CD8⁺ T cells, inguinal lymph nodes and spleens were harvested from 8-week-old female OT-1 transgenic mice (Jackson Laboratory). Single-cell suspensions were made from each tissue and pooled to increase total cell yields from each animal. Negative selection by a magnetic-activated cell sorting (MACs) kit (Miltenyi Biotec, Bergisch Gladbach, Germany) was used to purify CD3⁺CD8α⁺ T cells following the manufacturer’s protocol. Briefly, cells were incubated with biotin-antibody cocktail at 1 x 10⁷ cells / 50 μL total volume (40 μL buffer + 10 μL antibodies) for 5 minutes at 4°C. Labeled cells were then applied to an LS column (Miltenyi Biotec) in a QuadroMACs (Miltenyi Biotec) magnetic separator. Unbound cell fraction, containing CD3⁺CD8α⁺ cells was collected and counted. OT-1 T cells were then labeled with 5 μM CTV dye. 5 x 10⁵ CTV-labeled T cells were seeded in a 96-well round-bottom plate alone or with 5 x 10⁵ MPEK cell lines OVA, MC.OVA, or vector control. Co-cultures were incubated at 37°C in 5% CO₂ for 60 hours. After 60 hours, cells were harvested from wells and stained with anti-mouse-CD3-AlexaFluor647 (Cat# 100209, eBioscience, San Diego, CA), anti-mouse-CD8α-PE/Cy7 (Cat# 100722, BioLegend) and analyzed by flow cytometry. Live cells within the CD8⁺CD3⁺ gate were measured for proliferation by dilution of CTV dye.