Streptomycin penicillin

The human melanoma cell line (SK-MEL-28) was donated by Dr. Gustavo Jacob Lourenço from the University of Campinas, São Paulo, Brazil, and it was cultured at Dulbecco’s modified Eagle’s medium (DMEM) high glucose supplemented with 10% fetal calf serum (FCS) and 1% streptomycin/penicillin (Cultilab, Campinas, São Paulo, Brazil) at 37 °C in a 5% CO₂ atmosphere.
DPG [chemical abstracts service (CAS) number 68797-35-3] and 12-O-Tetradecanoylphorbol-13-acetate (TPA) (CAS number 16561-29-8) were obtained from Verdi Cosmetics LLC (Joanópolis, São Paulo, Brazil) and Sigma Chemical (St. Louis, MO, USA), respectively. For cell line treatments, DPG and TPA were diluted in DMEM to prepare a 2000 μM and 50 ng/μL stock solution, respectively. All treatment assays were performed in the presence of 10% FCS.

U87MG and T98G cell lines were gently donated by Dr. Adriana da Silva Santos Duarte from Hemocenter, State University of Campinas, Campinas, São Paulo, Brazil. Both were cultured in DMEM supplemented with 10% FCS and 1% streptomycin/penicillin (Cultilab, Campinas, São Paulo, Brazil). For all experiments, 1 × 10⁶ cells/ml were seeded and grown for 48–72 h before experimental treatments. Cells were maintained at a 37°C, 5% CO₂ environment and were passaged by Trypsin 0.25% (Cultilab) every 3–4 days. Cells were fed every 2–3 days and used for the experiments until the seventh passage after thawing.

Mice were euthanized 60 days after infection (i.e., 30 days after therapy with DCs or injection with PBS) and their lungs were removed. To analyze the fungal burden by colony forming units (CFU), sections of each lung were removed, weighed and homogenized in PBS. Aliquots of 100 μl were plated onto Brain Heart Infusion (BHI) agar supplemented with 5% spent supernatant from P. brasiliensis Pb192 cultures, 10 IU/ml streptomycin-penicillin (Cultilab, Brazil), 4% fetal bovine serum (Gibco), and 500 mg/ml cycloheximide (Sigma, St. Louis, MO, United States) (Moscardi-Bacchi et al., 1994 (link)). CFUs were determined after 10 days of incubation at 37°C.

Mice were euthanized 45 days after infection (day 65 of experiment) and lungs were removed. Portions of lung were weighed, homogenized in PBS and aliquots were plated on agar brain heart infusion (BHI) supplemented with 4% fetal bovine serum (Gibco, Grand Island, NY, United States), 5% supernatant of spent culture of P. brasiliensis Pb192, 10 IU/ml streptomycin-penicillin (Cultilab, Brazil) and 500 mg/ml cycloheximide (Sigma, St. Louis, MO, United States) (Moscardi-Bacchi et al., 1994 (link)). Plates were incubated at 37°C for 20 days, and the resulting Colony Forming Units (CFUs) were enumerated.

U87MG, T98G, U251, and U138MG were kindly donated by Dr. Adriana da Silva Santos Duarte, Hemocenter, University of Campinas, Campinas, São Paulo, Brazil, and were cultured at 37°C in Dulbecco’s modified Eagle’s medium (DMEM) high glucose supplemented with 10% fetal calf serum (FCS) and 1% penicillin/streptomycin (Cultilab, Campinas, São Paulo, Brazil) at 37°C in a 5% CO₂ atmosphere. For all experiments, the cells were seeded and grown for 72 h before the experimental treatments. The cells were passaged by Trypsin 0.25% (Cultilab) until the seventh passage after thawing.
DPG (chemical abstracts service number 68797-35-3) was obtained from Verdi Cosmetics LLC (Joanópolis, São Paulo, Brazil). For the cell line treatments, DPG was diluted in DMEM to prepare a 2,000-μM stock solution. All treatment assays were performed in the presence of 10% FCS and 1% P/S.

All compounds were subjected to the MTT colorimetric assay using Balb-c 3T3 clone A31 cells (mouse fibroblast cells) acquired from the Rio de Janeiro Cell Bank (BCRJ code 0047). Inhibition values were determined in the cell-based assay as previously described [46 (link)]. Briefly, cells were cultured at 37°C in an atmosphere of 5% CO₂ using DMEM medium (Cultilab, Campinas-SP, Brazil) supplemented with 3.5 g glucose (Sigma-Aldrich), 1% penicillin/streptomycin solution and 10% FBS (Cultilab). Cells were plated at a concentration of 10⁵ cell/mL in 96-well plates and incubated for 24 h. All compounds were freshly diluted from 50 mM DMSO stock solutions to obtain the final concentration of 250 μM and added to each well by replacing the medium. The cell viability was assessed during 24, 48 and 72 h using the MTT (Sigma-Aldrich) reagent, with an incubation time of 3 h. Formazan crystals were dissolved using a solubility reagent composed of DMSO, glacial acetic acid and extran for 1 h. The readout was obtained using Biotek Synergy HT plate reader at 570 nm. Benznidazole was used as control. This assay was done in quatruplicate in two independent experiments. Statistical analyses were made in GraphPad Prism 5. Dunnett’s multiple comparison tests were performed using benznidazole as the standard compound for the ordinary ANOVA analysis using 95% confidence interval.

E. globulus oil was supplied by Ferquima (São Paulo, Brazil). Sorbitan monooleate (Span 80®) was supplied by Sigma-Aldrich (São Paulo, Brazil). Polysorbate 80 (Tween 80®) was supplied by LabSynth® (São Paulo, Brazil). Caprylic/capric triglyceride mixture was acquired from Alpha Química Ltda. (Porto Alegre, Brazil). Histopaque was supplied by Sigma-Aldrich (São Paulo, Brazil). Fetal Bovine Serum, RPMI 1640, and Penicillin/Streptomycin were supplied by Cultilab (São Paulo, Brazil). Dimethyl sulfoxide (DMSO) and trypan blue were supplied by Nuclear (São Paulo, Brazil).