Alexa fluro 488 annexin 5 dead cell apoptosis kit

hBMMSCs were cultured in high glucose DMEM medium with supplements using 6-well plates. As the cells completed 60% confluence, they were grouped and treated for 72 h with 100 nM RvE1 + 1 μg/mL LPS, 100 nM MaR1+ 1 μg/mL LPS, and a combination of 100 nM RvE1 + 100 nM MaR1 + 1 μg/mL of LPS. Detached and adherent cells were harvested, centrifuged, and then stained with propidium iodide (PI) and Alexa Fluro 488 Annexin V using Alexa Fluro 488 Annexin V/Dead Cell Apoptosis Kit (Molecular Probes, Life Technologies, Eugene, Ore, USA). Stained cells were then analyzed by flow cytometry and BD FACSDiva Software (BD Biosciences, Franklin Lakes, NJ, USA). The experiment was carried out three times (n = 3) to confirm the findings.

Resolvin E1 (RvE1) and Maresin1 (MaR1) specialized synthetic pro-resolving inflammatory mediators were commercially purchased from GLPBIO Technology, Montclair, CA, USA. Stock solutions (10 µM) of each were prepared in absolute ethanol and stored at −80 °C for further use. Real-Time Cell Analyzer (RTCA) (ACEA Biosciences, Santa Clara, CA, USA) was used to investigate the proliferation and migration rates under different concentrations (10, 50, 100, 150 nM) of both mediators. In addition, to assess the apoptotic effect of these agents, treated cells were stained with annexin V/PI using Alexa Fluro 488 Annexin V/Dead Cell Apoptosis Kit (Molecular Probes, Life Technologies, Eugene, Ore, USA) and analyzed using BD LSR Flow Cytometer and BD FACSDiva Software (BD Biosciences, Franklin Lakes, NJ, USA). The selected doses were based on previously reported dose–response studies [15 (link),16 (link)] with slight modifications according to our experimental optimization. Thereafter, 100 nM concentration was selected for both mediators RvE1 and MaR1 for further experiments.

Apoptotic and necrotic cells were assessed using Alexa Fluro 488 Annexin V/Dead Cell apoptosis kit (Thermo Fisher Scientific). Staining was performed according to manufacturer’s instructions.