N 2 hydroxyethylpiperazine n 2 ethane sulfonic acid hepes

Dulbecco’s modified Eagle’s medium (DMEM), Ham F12 culture medium, d(+)-glucose, l-glutamine, N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES), fetal bovine serum (FBS), and the antibiotic-antimycotic solution, were obtained from Life Technologies (Grand Island, NY, USA). H89, chelerythrine, fluorescein diacetate-acetoxymethyl (FDA-AM), dimethyl sulfoxide (DMSO), insulin, Triton X-100, and bovine Hb were purchased from Sigma-Aldrich (St. Louis, MO, USA). The lactate dehydrogenase (LDH; EC 1.1.1.27) assay kit was obtained from Bio-Maghreb (Tunis, Tunisia). 5-6-chloromethyl 2′-7′dichlorodihydrofluorescein diacetate (CM-H₂DCFDA), JC-10, and 4,5-diamino fluorescein diacetate (DAF-FM) were from Molecular Probes (Eugene, OR, USA). U0126 and Apo-ONE Homogeneous Caspase-3/7 assay kit were supplied by Promega (Charbonnières, France).

C57BL/6-CD45.1 mouse BM cells, unfractionated or following magnetic c-Kit⁺ cell enrichment, were cultured using Ham’s F-12 medium (Wako), supplemented with 0.1% PVA (Sigma, Cat# P8136), 1% Insulin-Transferrin-Selenium-Ethanolamine (ITS-X) (100X) (Thermo Fisher Scientific), 1% Penicillin-Streptomycin-L-Glutamine Solution (100X) (Wako), N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES) (10 mM; Gibco), mouse TPO (100 ng/ml; PeproTech), and mouse SCF (10 ng/ml; PeproTech) for 28 days, incubated at 37 °C in a humidified 5% CO₂ incubator. Medium was changed every other day^{15 (link),16 (link)}. Magnetic cell separation was performed using anti-mouse c-Kit MicroBeads (Miltenyi Biotech, Cat# 130-091-224) according to the manufacturer’s instructions. Cell culture using either commercially available pre-packaged HemEx-Type9A (NIPRO) or in-house prepared medium supplemented with 100 ng/mL mouse TPO and 10 ng/mL mouse SCF. Unfractionated whole BM cells were seeded at 2 × 10⁶/mL onto 100 mm dish in 10 mL culture medium (day 0–14) or 60 mm dish in 4 mL culture medium (day 15–28). Magnetic column-enriched c-Kit⁺ BM cells were seeded at 1 × 10⁶/well onto 48-well plates in 1 mL culture medium. For long-term cultures, complete medium changes were made every 2 days and cell cultures were passaged at a ratio of 1:2-3 when cells exhibited 80–90% confluency.