Streptomycin

Aedes aegypti (Red Eye strain) were raised in an insectary at the Federal University of Rio de Janeiro, Brazil, under a 12 h light/dark cycle at 28°C and 70–80% relative humidity. Larvae were fed with dog chow. Adults were maintained in cages and fed a solution of 10% sucrose ad libitum. Four- to seven-day-old females were used in the experiments. Aedes aegypti Aag-2 cells were maintained at 28°C in Schneider´s Drosophila medium with L-glutamine (Life Tecnologies, Grand Island, NY) supplemented with 10% Fetal Bovine Serum (FBS) (Cultilab, Campinas, SP) and penicillin (100 u/mL) and streptomycin (100 μg/mL) (LGC biotecnologia, Cotia, SP).

Mosquitoes were artificially fed with different diets: (1) 10% sucrose (ad libitum), (2) rabbit blood, (3) bicarbonate-buffered saline-agarose (BBSA) supplemented with Serratia marcescens (Sm). The BBSA solution was composed of glucose (10 mg), 500 mM freshly made bicarbonate buffer pH 7.4 (10 mL), 0.5 mg low melting-point agarose and 100 mM ATP, pH 7.4 (5 mL). The final volume was set to 500 mL with 150 mM NaCl. Feeding was performed using water-jacketed artificial feeders maintained at 37 ^o C and sealed with a Parafilm “M” (Sigma-Aldrich, St. Louis, MO) membrane. For depletion of mosquito’s microflora, females were fed with sucrose 10% supplemented with antibiotics, penicillin (100 u/mL) and streptomycin (100 μg/mL) (LGC biotecnologia, Cotia, SP) for 4 days as previously described23 (link).

Two human cell lines were chosen, representing in vitro models with a high (α/β)_x ratio (chondrosarcoma, SW1353) and a low (α/β)_x ratio (melanoma, SKMel). SW1353 (ATCC® HTB-94™, LGC Standards, Middlesex, UK) were cultured in DMEM/F12 (Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12), supplemented with 10% fetal calf serum, 25 mM HEPES and 100 U/ml penicillin and streptomycin. SKMel (ATCC® HTB-72™, LGC Standards, Middlesex, UK) cultivated in MEM (Minimum Essential Medium Eagle), supplemented with 10% fetal calf serum, 25 mM HEPES, 2 mM l-Glutamin and 100 U/ml penicillin and streptomycin (all GIBCO®, Invitrogen, Darmstadt, Germany). Both cell lines were cultured at 37 °C in a humidified atmosphere with 95% air and 5% CO2. Cells were seeded in chamber slide flasks (Nunc™ Lab-Tek™ II Chamber Slide™ System, Thermo Fisher Scientific, Waltham, MA, USA) with plastic slides at 2 × 105 cells per flask 24 h before irradiation. Immediately prior to the irradiation, the chamber slide flasks were filled air-bubble free with the respective non supplemented medium.

The mouse corticotrope tumor cell line AtT-20, secreting adrenocorticotropic hormone [30 (link),31 (link)], was kindly provided by Dr. Ulrich Renner and Prof. Dr. Günter K. Stalla from the Clinical Neuroendocrinology Group, Max Planck Institute of Psychiatry, Germany. The rat Wistar-Furth pituitary tumor cell line GH3, secreting growth hormone and prolactin [32 (link)], was obtained from the Cell Bank of Federal University of Rio de Janeiro, Rio de Janeiro, Brazil. The cell lines were maintained in Dulbecco’s modified culture medium that was supplemented with 10% fetal bovine serum, antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin, LGC Biotechnology, São Paulo, Brazil), and 5 mg/mL fungizone (amphotericin B) (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C and 5% CO₂.

The cerebellar granule (Cb) cell line derived from CD1 mice was a kind gift from Dr. Susan Cotman (MGH, Harvard Medical School, Boston, MA, USA) and maintained as described previously [23] (link). In short, the cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with d-glucose, L-glutamine and pyruvate, with the addition of foetal calf serum, penicillin, streptomycin, Geneticin (Life Technologies, Darmstadt, Germany), and potassium chloride (Sigma Aldrich, Munich, Germany). The cells were maintained in a humidified incubator set to 33°C under 5% CO₂ atmosphere. The HeLa cell line (LGC Standards GmbH, Wesel, Germany) was grown in DMEM supplemented with glucose, foetal calf serum, penicillin and streptomycin in a humidified cell incubator set to 37°C under 5% CO₂.

Glutaraldehyde (GA, 25%v/v aqueous solution), dimethyl sulfoxide (DMSO) and monobasic anhydrous potassium phosphate were purchased from Quimiobras Indústria Química (Masssaranduba, SC, Brazil). Sodium chloride, potassium chloride, and methanol came from ISOFAR Indústria e Comércio de Produtos Químicos (Duque de Caxias, RJ, Brazil). Amphotericin B came from Indofine Chemical Company (Hillsborough, NJ, USA). Brain heart infusion medium and Sabouraud Dextrose Agar were purchased from HiMedia Laboratories, LLC (Mumbai, India). Streptomycin and penicillin were purchased from LGC Biotecnologia (Cotia, SP, Brazil). Fetal bovine serum (FBS) was purchased from Gibco™ (Gaithersburg, MD, USA). Dibasic sodium phosphate, Schneiders modified medium, Dulbecco´s Modified Eagle´s Medium–High glucose (DMEM–HG), and anhydrous poly(vinyl alcohol) (PVA, 98% hydrolyzed, Mw 13.000–23.000 g/mol) were purchased from Sigma-Aldrich (St. Louis, MO, USA).