Anti cd45 fitc

After the kidney tissue was dissociated by Liberase TM (Roche) in RPMI-1640 medium at 37°C for 20 minutes, 100 μL of the single-cell suspensions was incubated with the corresponding antibodies at room temperature for 30 minutes in the dark. The following antibodies were purchased from BioLegend: FITC anti-CD45 (catalog 103108), APC/Cyanine7 anti-CD11b (catalog 101226), PE/Cyanine7 anti-F4/80 (catalog 123114), and APC anti-Ly6c2 (catalog 128016). PE anti-Arg-1 (catalog IC5868P) was purchased from R&D Systems. The data were analyzed by using Kaluza.

The stem cell surface markers of the MSCs were administered using flow cytometry, which was stained with a Fixable Viability Kit (Biolegend, San Diego, CA) and Mouse MSC Analysis Kit (Cyagen, Santa Clara, CA) for 30 min according to the instructions. The CFSE-labeled MSCs were harvested on the 2nd three-day cultivation and detected by flow cytometry, with additional staining using the Fixable Viability Kit. The spleen and kidney single-cell suspensions from experimental mice were first stained via fluorophore-conjugated antibodies, including BV510 Fixable Viability Kit (Biolegend, San Diego, CA), FITC anti-CD45 (Biolegend, San Diego, CA), PerCP/Cy5.5 anti-CD4 (Biolegend, San Diego, CA), APC anti-CD25 (Biolegend, San Diego, CA) for 30 min at 4 °C, follow by PE anti-Foxp3 (Biolegend, San Diego, CA), and PE/Cy7 anti-mTOR (Invitrogen, Carlsbad, CA) for 2 h at 4 °C. The CD4⁺T cells cultured in vitro were stained via BV510 Fixable Viability Kit, PerCP/Cy5.5 anti-CD4, and APC anti-CD25 antibodies for 30 min at 4 °C. The CD4⁺CD25⁺CD127^–Tregs sorted by fluorescence activated cell sorting (FACS) were stained via BV510 Fixable Viability Kit, PerCP/Cy5.5 anti-CD4, APC anti-CD25, and PE anti-CD127 (Biolegend, San Diego, CA) antibodies for 30 min at 4 °C.

BALF was centrifuged at 300g for 5 min and 4 ℃. The supernatant was stored at -80 °C. Neutrophils were identified as CD45⁺Ly6G⁺CD11b⁺ when the cell sediments were resuspended in PBS and stained with FITC-anti-CD45, PerCP/Cyanine5.5-Ly6G, and APC/Cyanine7-CD11b (BioLegend, San Diego, CA, USA) 27 (link). Total cells in the BALF were counted using a hemocytometer.

Cells were resuspended in 100 μl FCB, blocked with 0.5 μl TruStainFcX (anti-mouse CD16/32 FcR block, Biolegend) for 5 min at room temperature, and stained for 30 min at 4 °C with: FITC-anti-CD45 (1:1000, Biolegend), PE-Cy7-anti-CD4 (1:200, eBioscience), PerCP-Cy5.5-anti-CD8 (1:200, Biolegend), PE-anti-B220 (1:200, Biolegend), APC-anti-MHCII (1:200, eBioscience), and APC-eFluor780-anti-CD11b (1:200, eBioscience) (Supplemental Table 1). Cells were then fixed in 1% PFA for 1 h. Finally, cells were resuspended in 500 μl FCB, labelled with DAPI to exclude cell debris, and analyzed with a CytoFLEX S flow cytometer (Beckman-Coulter) equipped with the CytExpert software (Beckman-Coulter).