Penicillin streptomycin p s

MSCs were isolated from subcutaneous adipose tissue donated freely by lipoplasty surgery patients according to the regulations approved by the Universidade Federal de Minas Gerais's Research Ethics Committee (348/2016). Cell extraction was carried out as described by Zuk et al. [12 (link)]. Briefly, tissue samples were washed twice with phosphate-buffered saline (PBS, pH 7.4; Thermo Fisher Scientific, Waltham, MA, USA) to remove blood cells and incubated with 0.1% collagenase solution (Thermo Fisher Scientific) for 45 min at 37°C. After incubation, samples were centrifuged at 300 ×g for 7 min, the supernatant was discarded, and the remaining pellet was resuspended in Dulbecco's modified Eagle's medium (DMEM; cat. number: 12800-017, Thermo Fisher Scientific) containing 10% fetal bovine serum (FBS; Thermo Fisher Scientific) and 1% penicillin/streptomycin (PS; Sigma-Aldrich, St. Louis, MO, USA). Cells were transferred to culture flasks and kept in a humidified atmosphere of 95% air and 5% CO₂ at 37°C. Culture medium was changed every 3 days.

AMs were isolated as described previously (Koppe et al., 2012a (link)). Briefly, the lungs were lavaged multiple times with 2 mM EDTA PBS (Gibco, Cambridge, MA, United States) (final volume 5 ml). The lavages were centrifuged (300 ×g, 10 min, 4°C) and the cell pellets were resuspended in 1 ml of RPMI1640 (Gibco, Cambridge, MA, United States) with 10% FCS (GE Healthcare, Little Chalfant, GB), 1% glutamine (Glu) and 1% penicillin/streptomycin (P/S) (both Sigma–Aldrich, Darmstadt, Germany). The AMs were seeded into cell culture plates and incubated at 37°C for 2 h. Then the medium was changed and cells were incubated overnight. Two hours before stimulation, medium was changed again to RPMI1640 with 2% FCS and 1% Glu.

17β-estradiol (E2), 4-nitrophenyl phosphate, diethanolamine, fulvestrant (ICI 182,780), 8-penylnaringenin (8PN), dicofol (Dic), endosulfan (End), fenarimol (Fen), glyphosate (Glp) and methiocarb (Met) were purchased from Sigma-Aldrich (Schnelldorf, Germany), xanthohumol (XN) and iso-xanthohumol (iX) from Extrasynthese (Genay, France). Concerning cell culture, flasks and dishes were obtained from Sarstedt (Nümbrecht, Germany), media (DMEM, DMEM/F-12) as well as fetal bovine serum (FBS) from GIBCO / Thermo Fisher Scientific (Karlsruhe, Germany) and penicillin/streptomycin (P/S) as well as carcoal dextrane-stripped FBS (CD-FBS) from Sigma-Aldrich (Schnelldorf, Germany).

CAKI-2 and 786-O RCC cell lines (European Collection of Authenticated Cell
Cultures, Public Health England, UK), both representative of ccRCC, were
cultured in RPMI medium supplemented with 10% foetal bovine serum (FBS) (Gibco,
UK) and 1% penicillin–streptomycin (P/S, 10,000 units of penicillin and 10 mg of
streptomycin/mL; Sigma, UK). Human umbilical vein endothelial cells (HUVECs;
PromoCell, Germany) were cultured in Endothelial Growth Medium supplemented with
1% P/S and 5% FBS and used up to passage 5. Human dermal fibroblasts (HDFs;
Lonza, UK) were grown in Dulbecco’s Modified Eagle Medium (DMEM; 4.5 g/L
glucose) with 10% FBS and 1% P/S. Cells were routinely maintained as 2D
monocultures in a humidified atmosphere at 37°C, 20% O₂,
5%CO₂.

Three days after intraperitoneal injection of 1–5% thioglycolate into the abdominal cavity of a nude mouse, peritoneal macrophages were obtained under sterile conditions. The cells were harvested by washing the peritoneal cavity with cold PBS (Gibco), then centrifuged (300 × g, 7 min) and resuspended in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (P/S) (Sigma-Aldrich, St. Louis, MO, USA). The cells were allowed to adhere for 4 h and then were washed to remove non-adherent cells and cultured in DMEM supplemented with 1% P/S. Finally, the purified macrophages were activated by incubating for 48 h with LPS (10 ng/mL; Sigma-Aldrich) or IL-4 (10 ng/mL; PeproTech).