High-purity primary podocyte cultures were obtained from the terminator (Podocin-Cre; Rosa-DTRflox) mice using a previously described method with slight modifications [48 (link)]. Briefly, mouse kidneys were cut into small pieces and incubated with 1 mg/mL type I collagenase (Sigma-Aldrich, Dorset, UK) at 37 °C for 45 min with occasional agitation; the cell suspension was then filtered through a 40-μm cell strainer (BD Biosciences, Oxford, UK) and seeded onto multiple rat tail collagen pre-coated plates. Forty-eight hours after seeding, medium containing diphtheria toxin (Sigma, St. Louis, MO, USA, 100 ng/mL) was applied to the culture for two weeks.
Immortalized mouse podocytes (MPC5, a generous gift from Peter Mundel, Boston, MA, USA) were cultured under growth-permissive conditions on rat tail collagen type I-coated plastic dishes (Corning, Franklin Lakes, NJ, USA) at 33 °C in RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Gibco BRL, Gaithersburg, MD, USA), 10 U/mL mouse recombinant γ-interferon (Sigma, St. Louis, MO, USA), 100 U/mL penicillin and 0.1 mg/mL streptomycin (Gibco BRL, Gaithersburg, MD, USA). Podocytes were grown in a flask and incubated at 37 °C under 5% CO2 for a minimum of 10–14 days to allow the cells to differentiate.
Immortalized mouse podocytes (MPC5, a generous gift from Peter Mundel, Boston, MA, USA) were cultured under growth-permissive conditions on rat tail collagen type I-coated plastic dishes (Corning, Franklin Lakes, NJ, USA) at 33 °C in RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Gibco BRL, Gaithersburg, MD, USA), 10 U/mL mouse recombinant γ-interferon (Sigma, St. Louis, MO, USA), 100 U/mL penicillin and 0.1 mg/mL streptomycin (Gibco BRL, Gaithersburg, MD, USA). Podocytes were grown in a flask and incubated at 37 °C under 5% CO2 for a minimum of 10–14 days to allow the cells to differentiate.
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