Cd8α cell isolation kit

TDLNs from control PBS and Pant mice were collected and CD11c⁺ cells were sorted using the CD11c Microbeads UltraPure mouse kit (Miltenyi Biotec). CD11c⁺ cells were incubated in a 96-well plate for 1 h at 37°C with or without the OVA peptide (SIINFEKL). T cells were isolated and purified from the spleens of transgenic OT-1 mice by negative selection using the CD8α⁺ cell Isolation Kit from Miltenyi Biotec, according to the manufacturer’s instructions, and stained with the Cell Trace Violet Cell Proliferation Kit (Invitrogen), according to the manufacturer’s instructions. CD11c⁺ cells and T cells were co-cultured (ratio of 1:10) for 3 d. Cells were stained with a fixable blue dead-cell staining kit (Invitrogen) and anti-mouse CD8 antibody (53-6.7, 55304 BD; PE-Cy5). T cell proliferation was assessed using flow cytometry.

Control DTA and Nav_1.8-DTA mice were infected with HSV-1-OVA-TK⁻ by flank scarification. After four days, the brachial and axillary draining LN were collected and CD11c⁺ cells were sorted with the CD11c Microbeads UltraPure mouse kit from Miltenyi Biotec, according to the manufacturer’s instructions. CD11c⁺ cells were incubated in a 96-well plate for 1 h at 37 °C with or without the OVA peptide (SIINFEKL). T cells were isolated and purified from the LN and spleens of transgenic OT-I mice by negative selection with the CD8α⁺ cell Isolation Kit from Miltenyi Biotec, according to the manufacturer’s instructions, and stained with the Cell Trace Violet Cell Proliferation Kit, according to the manufacturer’s instructions. CD11c⁺ cells and T cells were co-cultured (ratio of 1:10) for three days. Cells were stained with a fixable blue dead-cell staining kit (Invitrogen) and an anti-mouse CD8 antibody (PE-Cy5, 53-6.7, 55304 BD). T cell proliferation was assessed by flow cytometry.