Phenol red free α mem

Timelapse imaging of live-cells was used to study Bcl11b gene expression dynamics in single cells (Figures 2 and 4F, and Figure 2—figure supplement 1, Figure 4—figure supplements 1–2, and Video 1). To prepare for multi-day imaging, PDMS micromesh arrays (250 μm hole diameter, Microsurfaces, AU) containing small microwells that prevent seeded cells from migrating out of a single imaging field of view on 40x objective were adhered to 24-well glass-bottomed plates (Mattek, Ashland, MA). To prevent overcrowding in microwells and enable proper cell tracking, non-GFP expressing OP9-DL1, described in Kueh et al., 2016 (link), and sorted CFP+ DN2 progenitors were plated at appropriate densities to achieve ~8 cells/microwell and ~1 cell/microwell, respectively. Cells were cultured in standard medium using Phenol Red-free αMEM (Gibco) and supplemented with 5 ng/mL Flt-3L and 5 ng/mL IL-7.

RPTPμ-knock-out/LacZ knock-in mice of 8 weeks old were killed by cervical dislocation. Both femurs and tibiae were isolated, the ends were removed, and the bone marrow was obtained by flushing with 5 ml 10 % FCS in PBS. For one differentiation assay, cells of 2–3 mice were pooled, seeded at a density of 1.5 × 10⁶ cells/well in 12-well plates and cultured in phenol red-free α-MEM (Gibco) supplemented with 10 % (v/v) heat-inactivated FCS, penicillin/streptomycin, and 50 µg/ml ascorbic acid (BDH Prolabo, VWR International, Radnor, PA, USA). The bone marrow cells were cultured for 21 days, and medium was refreshed every 3–4 days. From day 11 of culture onward, the cultures were either supplemented with 10 mM β-glycerol phosphate (Sigma-Aldrich, St. Louis, MO, USA) alone or stimulated with BMP-4 (50 ng/ml; R&D systems, Minneapolis, MN, USA), BMP-6 (100 ng/ml; R&D systems) or Noggin (250 ng/µl; R&D systems) as well. At day 21, the culture medium was withdrawn and the cell layers were processed for β-galactosidase activity determination either by staining with X-gal or by an enzymatic assay using o-nitro-phenyl-β-D-galactopyranoside (ONPG; Sigma-Aldrich). Parallel cultures were washed twice with PBS, fixed in 3.7 % formaldehyde in PBS for 5 min and stained with 2 % alizarin red S solution (Sigma-Aldrich).

Osteogenic differentiation assays were performed on MSCs and SV-HFOs. 3000 cells/cm² (MSCs) or 9000 cells/cm² (SV-HFOs) were seeded in α-MEM in 12-well-plates. For MSCs, the medium was replaced after 24 h with complete osteogenic medium; DMEM High Glucose (Gibco) with 10% FCS, 1.5 μg/mL fungizone, 50 μg/mL gentamicin, 25 μg/mL ascorbic acid-2-phosphate, 10 mM β-glycerophosphate, and 0.1 μM dexamethasone. For SV-HFOs, medium was replaced after 48 h with osteogenic differentiation medium consisting of phenol-red free α-MEM (Gibco) supplemented with 20 mM HEPES (Sigma, MO, USA), streptomycin/penicillin, 1.8 mM CaCl₂·2H₂O (Sigma), 2% heat-inactivated charcoal-treated FCS, 0.1 μM dexamethasone and 10 mM β-glycerophosphate. To both cultures FST315 (28, 70, and 175 ng/ml) was added during each medium refreshment. The experiment was carried out until onset of mineralization, monitored by measuring calcium concentration in the culture supernatant. For biochemical analyses, medium was collected and cells were scraped from the culture dish in PBS containing 0.1% Triton X-100. Supernatants were stored at −80°C. Cell lysates were thawed and sonicated on ice in a sonifier cell disruptor (Soniprep 150, MSE, London, UK) or in a water-bath sonifier (Ultrasonic Cleaner CD-4800, Norville, UK) before analysis.

Calcium nitrate and diammonium hydrogen phosphate were purchased from SD Fine-Chem, India. Minimal essential media-alpha (α-MEM), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and trypsin–EDTA were all purchased from Sigma Chemical Co. (St. Louis, MO, USA). Fetal bovine serum (FBS) and phenol red free α-MEM were purchased from Gibco (Waltham, MA, USA). Antibiotic cocktail (penicillin and streptomycin) was purchased from Hi-Media, India. Rifampicin and Isoniazid were purchased from Merck (Darmstad, Germany). All other chemicals used were of analytical grade.