Annexin 5 fitc and 7 aad

After 3 days drug treatment, cell proliferation and apoptosis were determined using BrdU proliferation assay kit (Abcam) and Annexin V-FITC and 7-AAD (Beckman Coulter) as described in our previous study (Li et al., 2018 (link)).

The cytotoxicity of the DHE was evaluated according to the European Standard ISO 10993-5-2009^{22 (link)}. Balb/3T3 clone A31 cells (ATCC, U.S.A) were cultured in Dulbecco’s modified eagle medium (DMEM) High Glucose 4,5 g/L (ATCC, U.S.A.) supplemented with 10% bovine calf serum (BCS, ATCC) and 1% ATB/ATM. Cells were seeded at 15 000 cells/cm² in a 48 wells plate and incubated at 37°C for 24 h. DHE samples of 5 mm diameter were prepared with a biopsy puncher (Kai medical, Solingen, Germany) and incubated with 200 µL/well of extraction medium (same composition as the Balb/3T3 culture medium but supplemented with 2% BCS). At 24 h, the supernatant was used to replace the culture medium of the Balb/3T3 cells and culture medium of the control cells was renewed. Cells were cultured for 72 h and then detached by a trypsin treatment. Viability was evaluated by Annexin V-FITC and 7AAD staining (Beckman Coulter France, Villepinte, France) according to the manufacturer’s instructions. Results were analyzed by flow cytometry using the Attune™⁻NxT cytometer (ThermoFiscer scientific).