M4526

The osteoblastic cell line MC3T3-E1 (93021013, Sigma-Aldrich Chemie GmbH, Schelldorf, Germany) was maintained in α-modified minimal essential medium (α-MEM, M4526, Sigma-Aldrich Chemie GmbH, Schelldorf, Germany), supplemented with 10% fetal bovine serum (Biowest SAS, France), 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin solution. Cells were cultured in a humidified atmosphere containing 5% CO₂ at 37 °C. On reaching 80% confluence, cells were detached every 3–4 days using 0.05% (w/v) trypsin, 0.02% (w/v) EDTA solution and not used beyond passage 20.
For the experiments, MC3T3-E1 cells were removed from tissue culture dishes using a trypsin-EDTA. Trypsin digestion was terminated by the addition of completed medium and the harvested cells were centrifuged at 200 × g for 5 min. The cell pellet was resuspended in assay buffer (20 mM HEPES HBSS, pH 7.4) for serum-free conditions or in serum-containing cell culture medium.

CHO DG44 cells, obtained from Dr. Lawrence Chasin at Columbia University, were maintained in α-MEM medium with nucleotide (Sigma, M8042) supplemented with 10% fetal bovine serum (FBS), as previously described [8] (link), [14] (link). Plasmid DNA was transfected into cells using Lipofectamine 2000 reagent (Invitrogen) and Opti-MEM (Gibco). One day after transfection, 5 µg/ml of blasticidine was added to select for transformants, and the concentration was increased to 10 µg/ml on day 10. Usually, on day 23, the medium was replaced with α-MEM without nucleotide (Sigma, M4526) supplemented with 10% dialyzed FBS (Thermo Scientific). Methotrexate (Sigma) was dissolved in water and added to the culture at 5 or 50 nM. Cloning of cells was performed by limiting dilution.

Following dissection, spines were cut into small fragments and digested for 2 hours in medium (alpha-MEM (Sigma-Aldrich®, M4526)) containing 1% (v/v) antibiotic/ antimycotic solution and 0.1% (w/v) type II collagenase (Gibco®, 17101-015) in an orbital shaker at 37 °C; cell clusters were subsequently dissociated by incubation for 10 minutes at 37 °C in cell dissociation solution (Sigma®, C1419). Cells were washed twice in FACS buffer (0.5% BSA in 2 mM EDTA (Sigma-Aldrich®, T4174) in PBS, sieved through a 40 µm cell filter, and labelled with 0.3 μM Draq7® and 0.5 μg/mL of PE-conjugated anti-CD24 antibody (Beckman Coulter®, Cat: PN IM1428U) in FACS buffer for 10 minutes in the dark, at 4 °C. Samples were then washed FACS buffer, re-suspended in ice-cold PBS and immediately used for FACS (FACS Aria II; BD Biosciences). An isotype control (0.2 μg/mL IgG1, BD Pharminogen®, 550617) and Draq7® viability dye (0.3 μM) were used to establish gates for viable CD24⁺ and CD24⁻ cells. Viable CD24⁺ and CD24⁻ cells were sorted separately into microcentrifuge tubes containing 350 μL of RLT lysis buffer (RNeasy micro plus kit, Qiagen®) and RNA extracted immediately. No differences were detected in the forward and side scatter distribution of CD24⁺ and CD24⁻ events within each sample.