Tcmk 1 cells

Mouse renal tubular epithelial (TCMK-1) cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The TCMK-1 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Gibco, USA) that was supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco) in a 5% CO₂ humidified atmosphere at 37 °C.

TCMK-1 cells (American Type Culture Collection, Manassas, USA), mouse kidney epithelial cell line, were grown in Dulbecco’s modified Eagle’s medium (DMEM)/F-12 medium (Gibco, Carlsbad, USA) with 10% (v/v) fetal calf serum (Gibco), 2 mM L-glutamine (Gibco), 100 U/ml penicillin G, and 100 mg/ml streptomycin (Sigma, Dorset, UK) at 37°C in a 5% CO₂ humidified atmosphere.
TCMK-1 cells were cultured in serum-free DMEM/F-12 medium (Gibco, Carlsbad, USA) and stimulated with 200 μM H₂O₂ for 24 h. The cells were then fixed and subjected to ISEL and developed with 3,3′-diaminobenzidine (DAB) chromogen (Vector). Antiproperdin antibody (ab186834, Abcam) diluted 1:100 was applied followed by biotinylated goat–antirabbit IgG (1:300, BA-1000, Vector) and alkaline phosphatase streptavidin (1:200, SA-5100, Vector). Binding was detected by Fast Red (Sigma) and hematoxylin counterstaining. For the negative control, the primary antibody was substituted with normal rabbit IgG of same species (Merck Millipore) at same protein concentration.