2.4. Flow cytometry and cell sorting

The single‐cell suspensions preparation and flow cytometry analysis was administrated as described previously. 13 Briefly, kidneys were cut into 1‐2 mm³ pieces before placed in DMEM containing 100 mg/mL deoxyribonuclease (DNase) I (Roche) and 1 mg/mL collagenase IV (Sigma Aldrich) for 40 minutes at 37°C with intermittent agitation. The digested cell suspension was then passed through a 40‐μm cell strainer and washed with PBS twice.

For fluorescence‐activated cell sorting (FACS) analysis of kidney samples, single‐cell suspensions were incubated with bovine serum albumin (BSA) to block non‐specific binding and antibodies to CD45 (BD), MHC‐II (Novus), CD11c (Abcam), CD68 (Novus), CD11b (Novus) and CD103 (BD), as well as antibodies to natural killer (NK) cell, T cell and B cell lineages (lin): CD3 (Biolegend), T cell receptor (TCR)‐β (Biolegend), TCR‐γδ (Biolegend), CD19 (Santa) and CD49b (BD). When FACS sorting was performed on the digested kidney single‐cell suspension, cells were pregated on hematopoietic cells using anti‐CD45 antibody. Then, lineages (CD3/ CD19/CD49b/ TCR‐β/ TCR‐γδ) were used to exclude NK cells and lymphocytes, and 4’,6‐diamidino‐2‐phenylindole (DAPI) was used to exclude dead cells. Then after gated renal mononuclear phagocytes (rMPs) as lin⁻ MHCII⁺ cell subsets, Renal CD68⁻ CD11c⁺ (rMP1), CD68⁺ CD11c⁺ (rMP2), CD103⁺ CD11b⁻ (rMP3), CD103⁻ CD11b⁺ (rMP4) cell subsets and splenic CD8⁺ T cells were analysed or sorted. The sorted cells were then used for further analyzations.

Other antibodies used in another study include CD86 (BD), CD80 (Biolegend) and granzyme B (Abcam), as well as corresponding isotype controls. Cells were analysed on a FACS Aria machine (BD Biosciences).