Rpmi medium without phenol red

L. amazonensis (MHOM/BR/2000/MS501) is maintained by successive passages in female BALB/cAn mice and periodically reisolated from the popliteal lymph node. In vitro, promastigote forms were maintained in axenic culture in Schneider's Insect medium (Sigma-Aldrich, St. Louis, MO) supplemented with 10% inactivated fetal bovine serum (Cultilab), 100 U/mL penicillin and 10 μg/mL streptomycin (Sigma-Aldrich, St. Louis, MO) at 26ºC in a BOD incubator, for a maximum of 6 passages. The strain, originally isolated from a human visceral case, has been characterized by isoenzymes and RFLP [20 (link)].
Peritoneal macrophages were obtained by peritoneal lavage of BALB/cAn and C3H/He mice (ICTB/FIOCRUZ) 72 hours after 3% thioglycollate intraperitoneal injection. After the lavage, cells were diluted in RPMI medium without phenol red (Sigma-Aldrich, St. Louis, MO) supplemented with 10% fetal bovine serum (Cultilab, Campinas, Brazil), 200 mM L-glutamine, 100 U/mL penicillin and 10 μg/mL streptomycin (Sigma-Aldrich, St. Louis, MO), seeded in a culture dish or over coverslips and kept at 34°C in a humidified atmosphere with 5% of CO₂. The BALB/cAn animals will be referred to in the article as BALB/c.

The production of ROS was measured in A375 cells (10⁴ per well) grown for 24 h in a 96-well plate in RPMI medium without phenol red (Sigma Chemical Co., Darmstadt, Germany). Cells were then washed with PBS and loaded with 10 μM 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetyl ester (CM–H₂DCFDA) (Molecular Probes-Invitrogen, Eugene, OR) for 25 min, in the dark. Afterwards, cells were washed with PBS and incubated with increasing concentrations of tested complexes. The fluorescence increase was estimated utilizing the wavelengths of 485 nm (excitation) and 527 nm (emission) in a Fluoroskan Ascent FL (Labsystem, Finland) plate reader. Antimycin (3 μM, Sigma Chemical Co., Darmstadt, Germany), a potent inhibitor of Complex III in the electron transport chain, was used as the positive control.

LNCaP, VCaP and PC3 cells were purchased from ATCC (Manassas, United States). Cells were grown in RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with fetal bovine serum (FBS) (BioWest, South American origin), at 10% in 37 °C and 5% CO₂ atmosphere. RWPE-1 cell line was acquired from ATCC and was cultured in Keratinocyte Serum Free Medium (Lonza, Allendale, NJ, USA), PrSC cell line was purchased from LONZA and was cultured in Stromal Cell Basal Medium (LONZA). When cells were treated with DHT hormone, RPMI medium without phenol red (Sigma) supplemented with 5% of charcoal stripped FBS (LONZA) was used.