Accutase

All MEF cells were starved in DMEM without FCS and antibiotics for 2 h. After stimulation with Hy-IL-6, cells were detached from the cell culture dish with 1 ml Accutase (Biowest, Nuaillé, France Cat. No. L0950-100). For intracellular staining cells were fixed. Therefore, 100 µl of the cell suspension was mixed with 100 µl paraformaldehyde (4 %) and incubated at 37 °C for 10 min followed by centrifugation at 230 g, 4 °C for 5 min. Cell pellets were suspended in ice cold 90 % methanol and incubated on ice for 10 min. Subsequently, cells were washed twice with cold BSA-EDTA-Buffer (2 % BSA, 2 mM EDTA in PBS) and incubated with fluorophore-coupled antibodies (1:200) overnight. Cells were washed for two times in BSA-EDTA Buffer before FACS analysis. 10,000 individual cells were measured per condition. Flow Cytometry Data were recorded on a BD FACS Canto II equipped with 3 lasers (405 nm, 488 nm, 663 nm, Firmware Version 1.47) using FACS Diva (BD Biosciences), Version 6.1.3. Data were analysed using FlowJo (Treestar, Ashland, OR, USA), Version 10. FCS files were converted to Microsoft Excel files. For representation data from independent experiments as indicated in the figure legends were normalised to 100 % and pooled. Centre line of Box-and-whisker diagrams depicts median. Box limits are the 25 and 75 % quantiles. Whisker depict the 2.5 % and the 97,5 % quantiles.

ALDH activity was analyzed as previously described^{15 (link)}. Shortly, cells were treated for 4 days with WF, RT-WF and WF + RIBE fluids, detached with Accutase (BioWest, France) and subjected to ALDEFLUOR Assay based on manufacturer protocol. For each sample negative controls were incubated under the same conditions with diethylaminobenzaldehyde (DEAB).

Primary myoblasts were seeded in 10 cm diameter dishes (Sarstedt 83.3902) coated with Collagen I (Sigma C8919) at 30% confluency in proliferation medium, as above and left to attach for 2 hr before adding bromodeoxyuridine / 5-bromo-2'-deoxyuridine (BrDU, Invitrogen B23151) to a final concentration of 75 µM and left to proliferate for 6 hr. Cells were then rinsed with warm DPBS, detached using Accutase (Biowest L0950) and fixed using ice cold ethanol. Samples were left at –20 °C over-night and then analysed by flow cytometry. Fixed cells were submitted to an acid wash (2 M HCl, 0.5% Triton X-100) for 30 min at room temperature, washed in PBS and the remaining acid was neutralized using a borate buffer (pH = 8.5). Cells were then stained in 200 µL of PBS (0.5% Tween 20) with 1 µg of Anti-BrDU FITC conjugated antibody (BD 556028) or an FITC conjugated isotype control for 30 min at room temperature. Cells were then washed in HEPES buffer (pH = 7.4) and resuspended in PBS before processing using a FACScalibur system.

BMDMs were cultured under different HPs and types of stimulation for 2 or 7 days. For flow cytometric analysis, cells were treated with Brefeldin A (eBioscience) for 6 h and subsequently removed from culture dishes using Accutase (Biowest, Nuaille, France). Cells were then blocked with Fc block (2 μl/10⁶ cells, BD Biosciences, Hamburg, Germany) for 5 min at 4°C to avoid unspecific staining. To characterize macrophages, cells were stained using antibodies targeting F4/80, CD86, CD206, TNF-α, and IL-10 (27 (link)). Data were collected with a flow cytometer CytoFLEX (Beckman Coulter GmbH, Krefeld, Germany), and Kaluza Analysis software 2.1. (Beckman Coulter GmbH) was used for analysis.

SD rat embryos were obtained at 14.5 days. The cerebral cortex was harvested and immersed in DMEM/F12 (Gibco). The meninges were removed and washed in DMEM/F12 by centrifugation for 5 min at 500 × g. The pellet of the cerebral cortex was treated with Accutase (Biowest, Nuaillé, France). The Accutase digest was neutralized by adding DMEM/F12 containing 1% N2 supplements, 20 ng/mL EGF, 20 ng/mL FGF, and 1% antibiotics-antimycotics. Cells were then spread by pipetting up and down several times and pelleted by centrifugation. After re-suspension in the culture medium, the cells were transferred into T25 flasks and cultured in a 37 °C incubator. After 5–7 days of culture, the neurospheres were passaged to expand the NSCs for future experiments.

Cells were stimulated with wound fluids and conditioned medium and analysed at 9 time points: 30 min and 1, 2, 4, 8, 24, 48, 72 and 96 h after addition of fluids. Cells were then collected using Accutase (Biowest, France), fixated with BD Cytofix/Cytoperm™ Fixation/Permeabilization Solution (BD Biosciences, NJ, USA) and stained with fluorochrome-conjugated monoclonal antibodies: anti-human active caspase-3 antibody (Alexa Fluor 647 conjugated, rabbit IgG) (BD Biosciences, NJ, USA, Catalogue No. 552933), anti-human cleaved PARP antibody (PE conjugated, mouse IgG1) (BD Biosciences, NJ, USA Catalogue no. 552933) and anti-human γH2AX antibody (Alexa Fluor 488 conjugated, mouse IgG1) (BD Biosciences, NJ, USA Catalogue No. 560445). The stained cells were analysed using BD Accuri C6 (BD Biosciences, NJ, and USA). For quantification of each fluorescence signal, the median fluorescence intensity (MFI) was used. The results were normalized to the MFI of control (untreated) cells for each time point analysed.