Mem alpha medium

Pre-leukemic and leukemic thymocytes were purified by flow cytometry and co-cultured on GFP-positive OP9 and OP9-DL1 stromal cell lines, as described previously^{91 (link)}. Thymocytes were co-cultured in reconstituted alpha-MEM medium (#12318, Gibco, Grand Island NY, USA) supplemented with 20% FCS (#12318), HEPES 10 mM (#15630-080), sodium pyruvate 1 mM (#11360-070), β-mercaptoethanol 55 μM (#21985-023), Glutamax 2 mM (#15750-060), 1% Penicillin/Streptomycin (#15140-122), 5 ng/mL rmFLT-3 ligand (#250-31 L, Pepro Tech), 5 ng/mL rmIL-7 (#217-17, Pepro Tech) and 25 ng/mL rmSCF (#250-03, Pepro Tech). After 48 h of co-culture, thymocytes were counted and analysed by FACS.

Allo-mBM-MSCs were collected by flushing the femurs and tibias of 8-week-old C57BL/6 male mice with Alpha-MEM medium (Gibco, Auckland, NZ) followed by centrifugation. Cells were plated at a density of 1×10⁶ nucleated cells/cm² and cultured in Alpha-MEM supplemented with 10% fetal bovine serum (HyClone Laboratories, Logan Utha) and 80 μg/mL gentamicin (Sanderson Laboratory, Chile), cultures were maintained at 37°C in 5% CO2 and 95% air atmospheric condition. After 72 hours, non-adherent cells were removed and fresh medium was added to the cells.
Adherent cells were detached after 96 hours using 0.25% trypsin and 2.65 mM EDTA (Life Technologies, Grand Island, NY), then centrifugated and sub-cultured at 7,000 cells/cm². Allo-mBM-MSCs were injected after one subculture.

Cell culture experiments were performed for the scaffold revealing the best structural properties. Mouse calvarial preosteoblast cell line (MC3T3-E1 Subclone 4, ATCC-LGC, standards, Teddington, UK) was used to assess cytotoxicity of the fabricated bone scaffold and cell growth on its surface. MC3T3-E1 cells were cultured in alpha MEM medium (Gibco, Life technologies, Carlsbad, California, USA), supplemented with 10% fetal bovine serum (Pan-Biotech GmbH, Aidenbach, Bavaria, Germany), 100 μg/mL streptomycin (Sigma-Aldrich Chemicals, Warsaw, Poland), 100 U/mL penicillin (Sigma-Aldrich Chemicals, Warsaw, Poland), and maintained at 37 °C in 5% CO₂ in air atmosphere.

All reagents and HPLC solvents, unless indicated otherwise, were purchased from Sigma-Aldrich (St. Louis, MO). Monocalcium phosphate monohydrate (MCPM) was purchased from Scharlau (Barcelona, Spain). Citric acid was purchased from Amresco (Solon, OH). Rebamipide hydrate was purchased from Santa Cruz biotechnology (Santa Cruz, CA). MC3T3 mouse calivarial osteoblasts and Raw 264.7 mouse macrophages were purchased from American Type Cell Culture (ATCC, Manassas, VA)[59 , 60 (link)]. Raw cells were sub-cultured in Dulbecco’s modified eagles medium (DMEM/F12) with 10% heat inactivated fetal bovine serum (Fisher Scientific, Pittsburg, PA), 1 mg ml^-1 streptomyocin and 1,000 units ml^-1 of penicillin (Fisher), in 5% CO2 atmosphere at 37^°C. MC3T3 and Raw 264.7 were subcultured upon reaching 80% confluence and only fractions containing >95% viability (trypan blue) were used for further testing. MC3T3 were tested within 8 passages of receipt from ATCC for differentiation and proliferation testing. Raw macrophages were used within 16 passages of receipt from ATCC for proliferation and ROS testing. MC3T3 cells were grown in alpha MEM medium (Gibco BRL) for subculture and proliferation studies. For differentiation studies 10mM beta-glycerol phosphate and 50ug ml^-1 ascorbic acid was added to the proliferation media. Alkaline phosphatase substrate and microBCA kits were purchased from Sigma.

BMSCs were cultured from bone cavity of femurs and tibias of C57BL/6 mice. The cells were cultured with alpha-MEM medium (Gibco, USA) supplemented with 20 % fetal bovine serum (FBS, Gibco), 2-mM glutamine, 100-U/mL penicillin, and 100-mg/mL streptomycin (Invitrogen, USA), and then incubated in 5 % carbon dioxide at 37 °C. BMSCs between passages 3–4 was used in the following study.

Bone marrow cells obtained from the femurs and tibias of wild-type and knockout mice were cultured in alphaMEM medium (Gibco) containing 10 % fetal calf serum and 1 % penicillin-streptomycin. For in vitro osteoclast formation, we used mouse bone marrow cells stimulated in Petri dishes for 3 days with M-CSF 10 ng/ml (bone marrow macrophages; BMMs). BMMs were subsequently plated on 96-well plates and supplemented with 30 ng/ml M-CSF and 50 ng/ml RANKL with media/cytokine changes every 2 days. After 5–6 days under these osteoclastogenic conditions, we performed staining for tartrate-resistant acidic phosphatase (TRAP) and imaged the plates using an inverted microscope. The number of osteoclasts (OCs), defined as TRAP-positive cells containing ≥3 nuclei (TRAP-positive multinucleated cells), was counted manually in each well, while the number of cells containing <3 nuclei (TRAP-positive or TRAP-negative mononucleated cells) were counted in three power fields in each well and expressed as percent of control from at least three independent cultures. All agonists and antagonists were administered to BMMs together with M-CSF and RANKL unless otherwise noted.