100 um cell strainer

Mice were sacrificed, peripheral blood (PB) was collected, liver, spleen (SP), and mesenteric lymph nodes (LN) were removed. Spleen, and MLN were mechanically dissociated and processed through a 100-um cell strainer (BD Falcon), and suspended in HBSS. Lymphocytes were isolated by Ficoll-Hypaque (DAKEWE, SZ, China) density gradient centrifugation from cell solution and diluted in blood solution. Isolated cells were washed twice in HBSS and re-suspended at 2 × 10⁶ cells/ml in complete RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum (FCS), 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM glutamine, and 50 μM 2-mercaptoethanol.

Glomeruli from 8-week-old C57BL/6 mice were collected by filtering kidney tissues with different pore sizes mesh sieves. In brief, kidneys were cut into small pieces with a scalpel, immersed in 4 ml HBSS and treated with 1 mg/ml collagenase (Sigma, c6885) and 0.5 mg/ml pronase E (Sigma, p6911) at 37°C for 15 min. Then, tissues were filtrated through a 100-um cell strainer (BD Biosciences, San Jose, CA, United States) with a flattened pestle and rinsed with 15 ml HBSS. Afterward, the suspension was flown through a 400-mesh screen. Glomeruli retained on the screen were transferred with a pipette into a new 50-ml tube. Finally, the glomeruli were collected by centrifugation at 3,000 rpm for 10 min.
For primary podocyte culture, glomeruli were suspended with 1,640 containing 10% FBS and seeded on the culture dishes. The culture dishes were kept still for 3 days to allow the glomeruli to adhere. On day 4, culture medium was replaced with fresh one and the unattached glomeruli were washed away. On day 5, cellular outgrowths were detached with trypsin (Gibco) and filtrated through a 40-μm cell strainer to remove the remaining glomerular cores. The filtered cells were collected and seeded in 6-well plates at a density of 1.5×10⁵ at 37ºC with 5% CO₂.