Pe cy7 conjugated anti mouse cd8 antibody

OVA-specific CD8⁺ T cells harvested from transgenic OT-1 mice were used for the in vitro T-cell proliferation assay. Briefly, the lymph nodes and spleen of OT-1 mice were harvested, minced, and passed through a 70-μm pore filter. Then, CD8⁺ T cells were isolated using a MojoSort CD8⁺ T-cell isolation kit (BioLegend) according to the manufacturer’s protocol. The isolated OT-1 CD8⁺ T cells were stained with a CFSE cell division tracker kit (BioLegend) and cocultured with splenocytes (including DCs) isolated from wild-type C57BL/6 mice. Then, 1 μg/ml CCNVs and SCCNVs, which were produced from EL4 cancer cells or OVA-expressing E. G7-OVA cancer cells, was added to the cultures. An OVA epitope peptide (257-264, 1 mg/ml; ANASPEC, CA, USA) was used as the positive control. After 3 days of culture, the cells were harvested and stained with a BV421-conjugated anti-mouse CD3 antibody and PE-Cy7-conjugated anti-mouse CD8 antibody (BioLegend). Then, the percentage of CFSE^low CD3⁺ CD8⁺ T cells was calculated.

For FACS analysis of mouse renal cells, single-cell suspensions were stained with antibodies at 4 °C for 30 min. The following antibodies from Biolegend were used: BV421-conjugated Zombie (423114), APC/Cy7-conjugated anti-mouse CD45 antibody (103116), APC-conjugated anti-mouse CD3 antibody (100236), FITC-conjugated anti-mouse CD4 antibody (100406), PE/Cy7-conjugated anti-mouse CD8 antibody (100722), PE-conjugated anti-mouse B220 antibody (103208), PE/Cy7-conjugated anti-mouse CD31 antibody (102417), PE-conjugated anti-mouse GP38 antibody (127407).