Single-cell splenocyte suspensions were obtained by mashing total spleens through a 70-μm nylon cell strainer (BD), and red blood cells were lysed with a hypotonic ammonium chloride-potassium bicarbonate (ACK) buffer. Lymph nodes were homogenized by teasing between frosted glass slides followed by filtration through nylon cell strainer. IEL cells were isolated as previously described (Lefrancois and Lycke, 2001 ). Liver was homogenized and cells recovered after Percoll separation. Lungs and kidneys were cut in small pieces and digested in 1 mg/mL collagenase D and 0.1 mg/mL DNase I (Roche) for 2 hr at 37°C. Tissues were then homogenized and passed through a nylon strainer followed by an ACK lysis.
Transgenic naive iFx1f/f- and WT-P14 were isolated using the mouse CD8+ T cell enrichment kit (Miltenyi Biotech), and 103–105 P14 were transferred into naive CD45.1+ C57BL/6 mice. P14 cells were re-isolated from infected mice by staining total splenocytes in 10% FBS RPMI media with anti-CD8α (53-6.7), CD45.1 (A20), CD45.2 (104), and KLRG1 (2F1; all from ThermoFisher).
Transgenic naive iFx1f/f- and WT-P14 were isolated using the mouse CD8+ T cell enrichment kit (Miltenyi Biotech), and 103–105 P14 were transferred into naive CD45.1+ C57BL/6 mice. P14 cells were re-isolated from infected mice by staining total splenocytes in 10% FBS RPMI media with anti-CD8α (53-6.7), CD45.1 (A20), CD45.2 (104), and KLRG1 (2F1; all from ThermoFisher).