Dmem hpa

Single-cell suspensions of bone marrow were prepared by flushing the cells out from the femur and tibia using a syringe and a 21-gauge needle. After harvesting under sterile conditions, the spleens were passed through a wire mash and monodispersed in Dulbecco’s modified Eagle’s medium (DMEM-HPA, Capricorn Scientific, Germany) supplemented with 5% fetal calf serum (FBS, Biowes South America No. S1810). Then, 2 × 10⁵/mL bone marrow cells and 4 × 10⁵/mL splenocytes were separately plated in methylcellulose media (StemCell Technologies, Vancouver, BC, Canada) containing either 3 U/mL EPO (MethoCult M3334) or 3 U/mL EPO supplemented with 50 ng/mL SCF, 10 ng/mL IL-3, and 10 ng/mL IL-6 (MethoCult GF M3434). The cells were plated in duplicate in 35 mm tissue culture dishes (Sarstedt, Germany) and incubated at 37 °C in a humidified atmosphere containing 5% CO₂. According to the manufacturer’s instructions, burst-forming units-erythroid (BFU-E) were enumerated following the incubation period of 7 days in MethoCult GF M3434 medium, whereas colony-forming unit-erythroid (CFU-E)-derived colonies were scored after 2 days of culture in MethoCult M3334 using an inverted microscope.

Human umbilical vein endothelial cells (HUVECs) were purchased from PromoCell (PromoCell, C-12200). HUVECs were cultured in an endothelial cell growth medium kit (PromoCell, C-22110) with 1% penicillin/streptomycin (Capricorn Scientific, PS-B; Ebsdorfergrund, Germany). Vascular smooth muscle cells (VSMCs) were isolated from the thoracic aorta of a 7-week-old male Sprague-Dawley rat using previously published procedures [48 (link)]. Briefly, the rat was sacrificed with an overdose injection of Zoletil 50 and Rompun. Then, the rat was disinfected with povidone. The chest was opened and the thoracic aorta was dissected using micro-dissecting scissors to open the aorta and remove the endothelial layers by gentle scraping. The aorta was cut until 1–2 mm³ and treated with collagenase and elastase (Sigma, #E1250) for 2 h at 37 °C. The cells were transferred to a tube containing the cell culture medium and centrifuged. The cell pellets were washed with PBS 3 times. Finally, VSMC pellets were resuspended in the cell culture medium and cultured in a cell incubator. VSMCs were cultured in DMEM (Capricorn Scientific, DMEM-HPA; Ebsdorfergrund, Germany) with 10% FBS (Capricorn Scientific, FBS-11A; Ebsdorfergrund, Germany), 5% SMGS (Gibco, S-007-25), and 1% penicillin/streptomycin. All cells were cultured at 37 °C in a humidified environment containing 5% CO₂.

CCD-1137Sk human fibroblasts were kept in Iscove’s Modified Dulbecco’s Medium (Capricorn; IMDM-A; Lot #CP18-2245) supplemented with 10% fetal bovine serum (Capricorn; FBS-12B; Lot #CP16-1422), and 1% penicillin/streptomycin (Capricorn; PS-B; Lot #CP18-2207). HT-29 human colon cancer cells were cultivated in McCoy’s 5a media (Capricorn; MCC-A; Lot #CP19-2689), supplemented with 10% FBS-12B and 1% PS-B. The media for MDA-MB-231 human breast cancer cells consisted of Dulbecco’s Modified Eagle Medium (Capricorn; DMEM-HPA; Lot #CP18-2096) supplemented with 10% FBS-12B, 1% PS-B, and 1% Minimum Essential Medium Nonessential Amino Acids (Capricorn; NEAA-B; Lot #CP17-1726). All cell lines were passaged twice per week with a seeding density of 1 × 10⁶ cells/T75 flask. Two-dimensional cell culture analysis was based on cover slips (12 mm; VWR; ECN 631-1577; Lot #43395 819) placed in cell culture dishes (Greiner; PS; 664 160) before seeding a total of 1.2 × 10⁶ cells in appropriate ratios and culturing for four days.