Apc cy7 conjugated anti mouse cd45 antibody

The kidney tissue was minced with sterile scissors and digested with 1 mg/mL collagenase I for 45 min. Tissue suspension was terminated by digestion with PBS, filtrated by a strainer, and centrifuged at 1500 rpm for 5min. A single-cell suspension was prepared after red blood cell lysis. Cells were counted with a hemocytometer, and cell concentration was adjusted according to the count so that the cell suspension of each tube was 1 × 10⁶ cells. These samples were incubated with antibodies from Biolegend (San Diego, CA, USA) against BV510-conjugated Zombie, BV421-conjugated anti-mouse CD4 antibody, APC/Cy7-conjugated anti-mouse CD45 antibody, PE-conjugated anti-mouse PD1 antibody, PE/Cy7-conjugated anti-mouse CXCR5 antibody, BV605-conjugated anti-mouse ICOS antibody, PE-conjugated anti-mouse B220 antibody, Pecy5.5-conjugated anti-mouse IgD antibody, PE/Cy7-conjugated anti-mouse IgM antibody, FITC-conjugated anti-mouse CD38 antibody, APC-conjugated anti-mouse GL7 antibody, BV421-conjugated anti-mouse CD138 antibody. To detect TFH cells in peripheral blood leukocytes, we used the following antibodies from BD biosciences: KO525-conjugated anti-human CD4 antibody, FITC-conjugated anti-human CXCR5 antibody, PB450-conjugated anti-human PD1 antibody, PE/Cy7-conjugated anti-human ICOS antibody. Scheme of cell sorting for TFH and B lymphocyte subsets is presented in Figures S1 and S2.

For FACS analysis of mouse renal cells, single-cell suspensions were stained with antibodies at 4 °C for 30 min. The following antibodies from Biolegend were used: BV421-conjugated Zombie (423114), APC/Cy7-conjugated anti-mouse CD45 antibody (103116), APC-conjugated anti-mouse CD3 antibody (100236), FITC-conjugated anti-mouse CD4 antibody (100406), PE/Cy7-conjugated anti-mouse CD8 antibody (100722), PE-conjugated anti-mouse B220 antibody (103208), PE/Cy7-conjugated anti-mouse CD31 antibody (102417), PE-conjugated anti-mouse GP38 antibody (127407).

To analyze the frequency of OCP subsets, BM cells were collected from the mice, and washed three times with PBS supplemented with 4% FBS. After washing, the cells were treated with an anti-mouse CD16/CD32 antibody (BD, San Jose, CA) for 15 min on ice to block non-specific binding. The cells were then stained with APC-Cy7-conjugated anti-mouse CD45 antibody (Biolegend, San Diego, CA), FITC-conjugated anti-mouse CD117 antibody (Biolegend, San Diego, CA), PE-conjugated anti-mouse CD115 antibody (Biolegend, San Diego, CA), or APC-conjugated anti-CD11b antibody (Biolegend, San Diego, CA) for 30 min on ice. The cells were washed with PBS with 4% FBS, and analyzed using a Canto II (BD Biosciences, San Jose, CA) analyzer and Flowjo software to process the data (Tree Star, Ashland, OR). Monocytes/macrophages obtained from mouse bone marrow were cultured with M-CSF (30 ng/ml) for 3 days and treated with LPS (10 ng/ml) for 2 more days and were collected to analyze the RANK expression. APC-conjugated anti-mouse CD265 (RANK) antibody (Biolegend, San Diego, CA) was used to observe the RANK expression in OCPs.