Penicillin streptomycin antibiotic antimycotic solution abam

Spleen cell suspension was made by dissociation of spleen tissue with forceps. Single splenocyte was obtained by gently pipetting cell suspension. Afterward, red blood cells (RBC) were removed by adding RBC lysis buffer (Sigma, Saint Louis, MO, USA). Splenocytes were cultured in a plate at the density of 5–6 × 10⁶ in RPMI 1640 medium containing 10% fetal bovine serum (FBS; Sigma-Aldrich, Carlsbad, CA, USA) and 1% penicillin/streptomycin/antibiotic antimycotic solution (ABAM; Sigma-Aldrich, Carlsbad, CA, USA).

Hepatoma G2 (HepG2), NRK52E and Hela cells were purchased from ATCC (American Type Culture Collection, Manassas, VA), which were maintained in Dulbecco's modified Eagle's medium/Ham's F-12 medium (DMEM/F-12; GIBCO-BRL, Gaithersburg, MD, USA) supplemented with 5~10% fetal bovine serum (FBS; Sigma-Aldrich, Carlsbad, CA, USA) and 1% penicillin/streptomycin/antibiotic antimycotic solution (ABAM; Sigma-Aldrich, Carlsbad, CA, USA). For experiments, cells were exposed to stimuli in the absence of FBS.

Primarily, cultured urothelial cells were obtained from the bladders of C57BL/6J mice, using the method as previously reported 10. Briefly, the whole bladders were taken from anesthetized mice. The bladders were inverted by pushing the dome downward through the bladder neck with a blunt 18‐gauge needle and incubated with 0.05% trypsin‐EDTA for 30 min. at 37°C. After that, urothelial cells were collected, centrifuged and resuspended in keratinocyte serum‐free medium (KSFM, Invitrogen, Life Technologies, Carlsbad, CA, USA) containing 5 ng/ml epidermal growth factor, 50 μg/ml bovine pituitary extract, 30 ng/ml cholera toxin (all from Invitrogen) and 0.5% penicillin/streptomycin/antibiotic antimycotic solution (ABAM; Sigma‐Aldrich, Carlsbad, CA,USA). The cells were seeded in 96‐ or 24‐well plates precoated with collagen (0.01%; Sigma‐Aldrich) and cultured in a humidified atmosphere of 5% CO₂/95% air at 37°C. Cells after 72 hrs of cultivation were used for experiments.