Percp cy5.5 anti mouse f4 80

For surface markers, the cells were stained in PBS containing 1% BSA with indicated antibodies for 30 min on ice. For intracellular markers, the cells were first fixed with Fixation Buffer (420801; Biolegend, San Diego, CA, USA) at 4 °C for 30 min and then stained in Permeabilization Wash Buffer (421002; Biolegend, San Diego, CA, USA) with relevant antibodies at 4 °C for 30 min. Foxp3 staining was conducted according to the manufacturer’s instructions for the Mouse Foxp3 Buffer Set obtained from BD Bioscience (San Diego, CA, USA). The following antibodies were used for the studies: APC anti-mouse CD45 (103112), PE anti-mouse F4/80 (123110), PerCP/Cy5.5 anti-mouse F4/80 (123128), FITC anti-mouse CD11c (117306), APC anti-mouse CD206 (141708), APC anti-mouse/human CD45R/B220 (103211), PerCP/Cy5.5 anti-mouse Ly-6G/Ly-6C (108427), FITC anti-mouse CD4 (100406), PerCP anti-mouse CD8a (100732), AlexaFluor 647 anti-mouse/rat/human Foxp3 (320014), PE anti-mouse/human CD44 (103008), and APC anti-mouse CD62L (104412) from Biolegend (San Diego, CA, USA), and PE-Cy7 anti-mouse CD11b (552850) from BD Bioscience (San Diego, CA, USA). Flow cytometry data were acquired on MACSQuant^TM (Miltenyi Biotec, Auburn, CA, USA) and analyzed by FlowJo software (v10.5.3).

The cell suspensions were blocked with an Fc blocker (Anti-mouse CD16/32, 101319, Biolegend, USA) before incubation with surface marker antibodies. The following antibodies were used to stain cell surface markers: Briliant Violet 510 anti-mouse/human CD11b (101236, Biolegend, USA), CD206 Alexa 647 MR5D3 (565250, Biolegend, USA), and PerCP/Cy5.5 anti-mouse F4/80 (123127, Biolegend, USA). For the characterization of M2 macrophages, CD11b and F4/80 were used to define the population of macrophages. After that, CD206 was used to characterize the population of M2 macrophages in the total population of macrophages. The results were analyzed using FlowJo software.

Renal tissue was minced, digested by collagenase, washed with PBS, and finally prepared as a single cell suspension. The cells were then treated at 4 °C for 30 min in the dark with the following antibodies: APC anti-mouse CD11b (BioLegend, USA, 1:100), PE anti-mouse CD86 (BioLegend, USA, 1:50), FITC anti-mouse MHC-II (BioLegend, USA, 1:50), Percp/cy5.5 anti-mouse F4/80 (BioLegend, USA, 1:50), PE-cy7 anti-mouse Podoplanin (BioLegend, USA, 1:50). Flow cytometric analysis was performed using a BD FACSCanto II (BD, USA) and FlowJo software.