Flow cytometry cell sorter

As previously reported, Murine Aortic Endothelial Cells (MAECs) were isolated from mouse aorta [13 (link),67 (link)]. MAECs were selected by their ability to express the intercellular adhesion molecule-2 (ICAM-2) protein and purified with a flow cytometry cell sorter (DAKO). Purification was verified by confocal microscopy of MAECs double stained with von Willebrand factor antibodies.
MAEC were cultured with Dulbecco’s Modified Eagle’s Medium (DMEM/F12), supplemented with 0.05 mg/mL penicillin/streptomycin, 2.5 μg/mL amphotericin, and 10% Fetal Bovine Serum (Merck) in a humidified CO2 incubator with 5% CO2 at 37 °C. MAECs were used between passage 4 and 9.
BPA, BPS, and BPF concentrations between 100 and 500 µM were used to delimit the cytotoxicity on the MTT assay (and their respective proportions in the bisphenol mixture). Subsequently, the concentration range of 1 to 100 µM (1 + 0.38 + 0.36, 10 + 3.8 + 3.6, and 100 + 38 + 36 µM of BPA, BPF, and BPS, respectively) was used to explore cell adhesion and death.

Murine aortic ECs (MAECs) were isolated from mouse aorta, as previously reported^{31 (link)}. Briefly, the aortas were sectioned into 2-mm pieces, deposited in Matrigel solution, and fed with fresh growth medium for seven days [DMEM/HAM´s medium, 20% FBS, 0.05 mg/mL penicillin/streptomycin, and 2.5 μg/mL amphotericin]. The tissue was removed, and 500 μL of Cell recovery solution was added to each culture. The Matrigel layer was removed and poured on ice for one hour. The solution was centrifuged at 4 °C, resuspended in 4 mL of growing medium, and plated. MAECs were selected by their ability to express the intercellular adhesion molecule-2 (ICAM-2) protein and purified with a flow cytometry cell sorter (DAKO)³² . Purification was verified by confocal microscopy of MAECs double-stained with Von Willebrand factor antibodies.
H9c2 cells, a myoblastic cell line derived from embryonic rat myocardium, were cultured in the L-glutamine Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum and 0.05 mg/mL penicillin/streptomycin in a humidified CO₂ incubator with 5% CO2 at 37 °C^{33 (link)}.