Kidneys were harvested and cut into small fragments, and the tissue pieces were collected and dissociated with 1 mg/mL collagenase I (Gibco, USA) for 30 min at 37 °C. Then, the digestion solutions were filtered through 100 μm and 40 μm cell strainers (Falcon, USA) to obtain single-cell suspensions. Primary renal Mφ labeled with F4/80+ were sorted from mice with a FACSAria SORP cytometer (BD Biosciences, USA) and cultured with Mφ medium.
To analyze the phenotype of Mφ, flow cytometric identification was performed using combinations of the following mAbs: F4/80-APC (123116; Biolegend, USA), CD206-PE (141706; Biolegend), and CD11c-FITC (N418; Biolegend). After incubation for 30 min, flow cytometry analysis was performed on a CytoFLEX LX flow cytometer (Beckman Coulter, USA).
To detect the expression of LC3 in kidney-infiltrated Mφ (KM), the cells were blocked with Fc Block (BD Bioscience, USA) for 5 min and stained with the following antibodies: F4/80-PE (QA17A29; Biolegend) and LC3B (E5Q2K; CST). The concentration-matched mouse mAb IgG2b (E7Q5L; CST) was used as the isotype control. Alexa Fluor® 594-conjugated goat anti-mouse IgG (Poly4053; Biolegend) was used as a secondary antibody.
To analyze the phenotype of Mφ, flow cytometric identification was performed using combinations of the following mAbs: F4/80-APC (123116; Biolegend, USA), CD206-PE (141706; Biolegend), and CD11c-FITC (N418; Biolegend). After incubation for 30 min, flow cytometry analysis was performed on a CytoFLEX LX flow cytometer (Beckman Coulter, USA).
To detect the expression of LC3 in kidney-infiltrated Mφ (KM), the cells were blocked with Fc Block (BD Bioscience, USA) for 5 min and stained with the following antibodies: F4/80-PE (QA17A29; Biolegend) and LC3B (E5Q2K; CST). The concentration-matched mouse mAb IgG2b (E7Q5L; CST) was used as the isotype control. Alexa Fluor® 594-conjugated goat anti-mouse IgG (Poly4053; Biolegend) was used as a secondary antibody.