Nk1.1 pe cy7

Splenocytes and thymic cells were isolated from WT and TRPV5 KO mice. Spleens were cut into small pieces and incubated in RPMI with collagenase (1 mg/ml, Roche) and DNase (0.1 mg/ml, Sigma) at 37°C with occasional stirring for 20 min to extract various innate immune cells. Single-cell suspensions were obtained and incubated with various antibodies to distinguish cell populations for 1 h at 4°C and washed three times with PBS. Splenocytes and thymic cells were immunostained with CD4–PE (1 µg/ml, Biolegend), CD8–APC (1 µg/ml, Biolegend), and CD3–Fluorescein isothiocyanate (FITC) (2.5 µg/ml, Invitrogen) to compare T-cell populations. Innate immune cell phenotyping of splenocytes consisted of immunostaining with CD11b–PB (2.5 µg/ml, Biolegend), CD11c–APC (1 µg/ml, Biolegend), CD64–PE (1 µg/ml, Biolegend), F4/80-APC/Cy7 (1 µg/ml, Biolegend), MHC Class II–BV605 (1 µg/ml, Biolegend), LY6C–AF700 (2 µg/ml, Biolegend), GR1–FITC (2 µg/ml, Biolegend), and NK1.1–PE/Cy7 (1 µg/ml, Biolegend). Cells were fixed after washing in 4% paraformaldehyde for 10 min at 4°C followed by three washes with PBS. Cells were analyzed on an LSR Fortessa, and data were analyzed using FlowJo (BD).

For flow cytometric analysis of surface markers and intracellular cytokines, single cell suspensions of lungs, spleens, or cell cultures were stained with optimal concentrations of the following specific antibodies: CD45-V450, CD4-V500, CD8-V450, and CD62L-APC from BD Biosciences, CD3-PerCP-Cy5.5, CD4-BV510, CD4-PE-Cy7, CD8a-FITC, CD44-FITC, CD19-PE, CD80-AF488, CD86-APC, Ly6G-APC-Cy7, CD11c-PE-Cy7, NK1.1-PE-Cy7, TNFα-Pacific Blue, IFNγ-PerCP-Cy5.5, IL-17A-PerCP-Cy5.5, IL-2-PE-Cy7, and IL-10-PE from BioLegend and CD90.2-eFluor780 from eBioscience. Data were acquired on a FacsCantoII^® flow cytometer (BD Biosciences) equipped with a 405, 488, and 633 nm laser and analyzed with the FCS Express software (DeNovo™ Software).