Lsrii fortessa x 20 flow cytometer

C57BL/6 mice were injected intravenously with 10⁷actA-deficient Listeria monocytogenes expressing 2W protein25 (link), and on the same day injected intravenously with 500 μg of W6 (anti-2W:IA^b) blocking antibody. Seven days later, splenocytes were isolated and stained as above and magnetically enriched for 2W:IA^b -PE tetramer. Wild type 129S1 mice were infected with Salmonella Typhimurium expressing 2W, and 14 days following infection mice were treated intravenously with 500μg of blocking W6 MAb. Thirty-five days post infection, the spleen was removed and made into a single cell suspension and 10% was plated for bacterial CFU with the addition of 0.1% Triton 27 (link)(ref. 27). The remaining cell suspension was used for enrichment of 2W specific cells as described above. Cells were run on a LSRII Fortessa X-20 flow cytometer (Becton Dickinson) and analyzed using FlowJo software (v10).

Purified monoclonal antibody was directly conjugated to Alexa Fluor-488 by protein labelling kit (ThermoFisher Scientific) according to the manufacturer's protocol. Splenocytes were isolated from NOD mice, red blood cells lysed and resuspended at in complete DMEM at 6 × 10⁶cells ml⁻¹. p63 or OVA_141–160 was added to the media at a final concentration of 40 μM and the cells were incubated for 1.5 h at 37 °C with 5% CO₂. Cells were collected incubated in 2.4G2 for 10 min on ice and stained at 1:100 dilution for 30 min at 4 °C with FS1-AF488 and antibodies against CD4-BUV395 (GK1.5, BD Biosciences), CD8α-APC-ef780 (53–6.7, eBioscience), CD3ɛ-BV650 (145-2C11, BD Biosciences), B220-PE (RA3-6B, Tonbo), CD11c-PE-Cy (N418, eBioscience), CD11b-PerCp-Cy5.5 (M1/70, Tonbo), F4/80-APC (BM8.1, Tonbo), IA^g7-biotin (10.2-16, BioXcell), SA-BV421 (BioLegend) and dead cells were gated out using Ghost violet 510 viability dye (Tonbo), and run on a LSRII Fortessa X-20 flow cytometer (Becton Dickinson) and analyzed using FlowJo software (v10).