Facscanto 2 device

Human MSC obtained from dental pulp were purchased from Inbiomed (Inbiobank, San Sebastian, Spain). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)-low glucose (Gibco, Life Technologies) supplemented with 10% fetal bovine serum (FBS; Gibco, Life Technologies) and 1% penicillin/streptomycin (Gibco) in a humidified atmosphere of 95% air and 5% CO₂ at 37ºC.
Overexpression of HIF-1α was performed as previously described [22 (link)]. Briefly, MSC were transduced with a control pWPI-green fluorescent protein lentivirus (MSC wild type or MSC WT) or a HIF-1α-overexpressing pWPI-HIF-1α-GFP lentivirus (HIF-MSC) (http://addgene.org cat. #12254) daily for 3 days. Transduction efficiency was evaluated by flow cytometry (Coulter EPICS XL flow cytometer; Beckman Coulter) to determine the percentage of GFP-positive cells. The percentages of infection obtained were higher than 90% in all cases (Additional file 2: Figure S1). Cells were expanded and then cryopreserved until used.
For immunophenotypic characterization, MSC were incubated with the corresponding monoclonal antibodies (see Additional file 1). Then, they were acquired on a FACS Canto II device (BD Biosciences San Jose, CA) using the FACSDiva 6.1 software (BD Biosciences) and analyzed with the Infinicyt software.

For the T cell proliferation assay, syngeneic CD4⁺ /CD8⁺ T cells were isolated from spleens and lymph nodes of healthy mice and purified via MACS (Miltenyi Biotec, Bergisch Gladbach, Germany). The purity of CD4 and CD8 sorting was >98%. T cells were stained with 0.5 µM carboxyfluorescein succinimidyl ester (CFSE) for 10 min at 37 °C followed by thorough washing and resuspension in T cell medium. Five µg/ml Concanavalin A or 96-well plates pre-coated with 5 µg/ml anti-CD3 (clone 2C11) plus 5 µg/ml anti-CD28 (clone 37.51E1; both BD Pharmingen) served for T cell stimulation as indicated. Stimulated CFSE-labeled T cells were cultured in the presence or absence or either MSC-2 cells (pre-treated with 100 µg/ml mitomycin C in PBS for 10 min at 37 °C in a 5% CO₂ atmosphere) or bone marrow-derived myeloid cells in various ratios as indicated. After 72 h, the cultured cells were washed and gated on lymphocytes. The CFSE dilution due to cell division was analyzed by a FACSCANTO II device (BD Bioscience) using the FlowJo software (Tree Star).

Flow cytometry analysis was performed with C17.2 cells on FACSCANTO II device (BD Biosciences). After hypoxic treatment, cells were fixed with ethanol for viability and cell cycle analysis. Cell viability was measured with 1 × 10⁶ cells using LIVE/DEAD Viability/Cytotoxicity Kit (ThermoFisher Scientific). For cell cycle analysis, 1 × 10⁶ cells were analyzed with propidium iodide (Sigma).

CD34⁺ cells from 10 samples were collected after the co-culture with MSC WT or HIF-MSC, washed with PBS and incubated with the corresponding monoclonal antibodies (see Additional file 1). Then, they were acquired on a FACS Canto II device (BD Biosciences San Jose, CA) using the FACSDiva 6.1 software (BD Biosciences) and analyzed with the Infinicyt software. Data were represented as MFI.

The supernatants were tested for cytokines using the Legendplex^TM human inflammation 13-plex panel (Biolegend) for IL-1β, IFNα, IFNγ, TNFα, MCP-1, IL-6, IL-8, IL-10, IL-12p70, IL-17A, IL-18, IL-23, and IL-33. The minimum detectable concentration of each cytokine is given as 0.6–2.1 pg/mL by the manufacturer, but values less than 10 pg/mL were considered irrelevant. Samples were treated following the manufacturer’s instructions and measured with a FACS Canto II device (BD Biosciences). Analysis was done using Legendplex software (Biolegend). Supernatants were tested for type I Collagen using the MicroVue CICP EIA Kit for determining levels of CICP (C-Terminal of Type I Collagen; Quidel, San Diego, CA, USA) and CICP values were interpolated from the standard curve after measuring the optical density at 405 nm on a microplate reader (Bio-Rad, Munich, Germany).

After 72 h of co-culture, CD34⁺ cells were collected and washed. For apoptosis assays, cells were stained with Annexin V and 7-AAD using the BD Pharmigen PE Annexin V Apoptosis Detection Kit I (BD Biosciences). For cell cycle analysis, cells were stained with propidium iodide using the kit FxCycletm PI/RNase Staining Solution (Life Technologies), according to manufacturer’s instructions. Samples were acquired on a FACS Canto II device (BD Biosciences San Jose, CA) using the FACSDiva 6.1 software (BD Biosciences). At least 1.5 × 10⁵ events were recorded. For apoptosis assays, data were analyzed using Infinicyt (Cytognos). Cells were classified as early apoptotic, late apoptosis or dead if they were Annexin V⁺/7-AAD⁻, Annexin V⁺/7-AAD⁺ or Annexin V⁻/7-AAD⁺, respectively, as previously reported [2 (link)]. Ten samples were used for these experiments. For cell cycle analysis, ModFit LT version 5.0.9 (Verity Software) was used. Seven samples were analyzed. Data are represented as percentage of cells in each cell cycle phase.