I control microplate reader software

Cell viability was assessed using the MTT assay. To this end, mouse fibroblast cells (3T3) were grown in MEM, supplemented with 10% (v/v) FBS and 1% PSA. Then, the cells were seeded in a 96-well microplate at a cellular density of 1 × 10⁴ cells/well and cultured overnight. After MEM removal, CD-A (1.25 to 5 μM) or P/D/CD-A scaffold release product, MEM (negative control, PC), or 10% DMSO (positive control, NC) were added to the wells. After 24 h incubation in cell culture conditions, the solutions were discarded and the MTT assay was performed. In this regard, the MTT solution (200 μL, 0.5 mg/mL) was added to the cells and incubated for 3 h at 37 °C. Then, 200 μL of isopropanol was added for 20 min. Next, the absorbance was recorded at 570 nm (i-controlTM microplate reader software, TECAN Männedorf, Switzerland). The samples were considered non-toxic if cellular viability was higher than 70% compared to NC (based on the ISO 10993:2009 1-12 regarding the biological evaluation of medical devices).

Cell viability was assessed using MTT assay. Briefly, HUVEC density of 2.10⁴ cells/well was seeded and cultured overnight. Cells were treated with different samples: CD-NA and CD-SA (0–5 μM), any antioxidant (culture medium MEM, negative toxicity control, NTC), or 10% DMSO (positive toxicity control, PTC) during 24 h or 48 h. Afterwards, all solutions were washed and cells were incubated during 3 h at 37°C with 200 μL of MTT solution (0.5 mg/mL). Then, all wells were washed out of MTT solution and 200 μL of isopropanol were added for 20 min to solubilize the formazan crystals. The optical density was recorded at 490 nm (i-control microplate reader software, TECAN Männedorf, Switzerland). Not cytotoxicity of samples was considered if cellular viability was >70% of the control (based on the ISO 10993 : 2009 regarding the biological evaluation of medical devises).

C11-BODIPY581/591 is a fluorescent fatty acid analogue which allows the quantification of lipid peroxidation by indirect measure of mitochondrial ROS production [43 (link)]. Upon free radical-induced oxidation, its fluorescent properties shift from red to green [44 (link)]. Ninety-six well plates containing the cells were washed with PBS, before addition of C11-BODIPY (5 μM, during 30 min). Cells were incubated with 5 μM of CD-NA and CD-SA for 1 h, under light protection. Lipid peroxidation was initiated by addition of Cumene hydroperoxide (CumOOH, 50 μM). Fluoresce was monitored at red λex/λem = 590/7, 632/45 nm and green λex/λem = 485/14, 520/10 nm (i-control microplate reader software, TECAN Männedorf, Switzerland). Percent of CD-A cellular lipid peroxide activity inhibition was calculated relative to the positive control fluorescence intensity (PC; CumOOH without antioxidant).