Red blood cell lysis buffer

Podocyte and tubular cell isolation from mouse kidneys were performed as previously described with slight modifications^{51 (link),52 (link)}. In brief, podocytes were ex vivo selected by biotin-labeled anti-Kirrel3 and podocalyxin antibodies (R&D Systems BAF4910 and R&D Systems BAF1556, respectively), then isolated using magnetic, streptavidin labeled Dynabeads (Thermo Fisher). To isolate tubular cells, dissected and minced kidneys were digested with collagenase type II in RPMI media for 30 mins at 37°C. Cells were sieved first through a 100 μm nylon mesh, then through a 40 μm nylon mesh, followed by centrifugation at 500 g for 10 min. The pellet was resuspended in red blood cell lysis buffer (R&D Systems) and incubated on ice for 10min. After centrifuge at 500 g for 10 min, cells were resuspended in RIPA buffer (TEKnova) containing 1% protease inhibitor cocktail (Sigma) and stored at −80°C freezer for experiments.

Mouse spleens were harvested and washed twice with ice-cold phosphate-buffered saline (PBS). Spleens were grinded in cold PBS containing 2% FBS and filtered with a 200 mesh strainer. Cells were washed in PBS containing 2% FBS by centrifugation at 1200 rpm at 4°C for 10 min and the supernatants were discarded. After resuspension in 1 mL PBS containing 2% FBS, 100 μL aliquots of cell suspension were added to the appropriate tubes. Monoclonal antibodies (CD3-FITC, CD8-PerCP, and CD4-PE, 10 μL for each) were added along with appropriate isotypic negative. These tubes were incubated for 10 min at 4°C. Then 0.5 mL Red Blood Cell Lysis Buffer (R&D systems, Minneapolis, MN, USA) was added in the tubes and the cells were centrifuged at 1200 rpm for 10 min. The cells were washed with 1 mL PBS, centrifuged at rpm for 10 min, and resuspended in 0.2 mL PBS. For each sample, 10000 events were analyzed and the proportions of CD3+CD4+ and CD3+CD8+ splenic lymphocytes were determined by flow cytometry and CD3+CD4+ T cells were isolated for the next experiments. The ratio of CD4+/CD8+ T cells was then calculated.

Podocyte and tubular cell isolation from mouse kidneys were performed as previously described with slight modifications^{50 (link),51 (link)}. In brief, podocytes were ex vivo selected by biotin-labeled anti-Kirrel3 and podocalyxin antibodies (R&D Systems BAF4910 and R&D Systems BAF1556, respectively), then further purified using magnetic, streptavidin conjugated Dynabeads (Thermo Fisher). To isolate tubular cells, dissected and minced kidneys were digested with collagenase type II in RPMI media for 30 min at 37 °C. Cells were sieved first through a 100 μm nylon mesh, then through a 40 μm nylon mesh, followed by centrifugation at 500 g for 10 min. The pellet was resuspended in red blood cell lysis buffer (R&D Systems) and incubated on ice for 10 min. After centrifuge at 500 g for 10 min, cells were resuspended in RIPA buffer (TEKnova) containing 1% protease inhibitor cocktail (Sigma) and stored at -80^oC freezer for experiments.