Glomax multi

The viability of TM3 Leydig cells was evaluated using alamarBlue reagent (ThermoFisher Scientific, Invitrogen, Vantaa, Finland), as reported previously [27 (link),35 (link),36 (link)]. AlamarBlue assay is a sensitive oxidation-reduction indicator that fluoresces after the reduction of the blue color of resazurin to the pink color of resorufin, due to mitochondrial dehydrogenase activity in living cells [37 (link)]. In brief, Leydig cells were pre-cultured at an adjusted density (4 × 103 cells/well) in gelatin pre-coated 96-microwell plates, 24 h before the exposure. Afterward, the cell culture medium was removed, and fresh medium containing experimental doses (62.5–2000 µg/mL) of Lepidium sativum L. was applied for 24 h and 48 h. After the respective treatments, cells washed with Dulbeccos’s phosphate-buffered saline (DPBS; Sigma-Aldrich, St. Louis, MO, USA) were incubated with DMEM/F12 media (without phenol red) containing alamarBlue reagent in a final concentration of 5% (v/v). After 0.5 h min incubation in a CO₂ incubator (37 °C; 5% CO₂; and 95% atmospheric humidity), the fluorescence (excitation/emission: 530/590 nm wavelengths) was measured using a combined spectro-fluoro-luminometer GlomaxMulti+ (Promega Corporation, Madison, WI, USA).

Viability of coated MIN-6 cells using different coating deposition sequence was evaluated using a live/dead staining kit. Briefly on the day of experimentation, 1 μL of calcein acetoxymethylester (Calcein AM (662 Da), indicator for live cells, Invitrogen, USA) and 5 μL of propidium iodide (PI (668 Da), indicator for dead cells, Invitrogen, UK) were added to a 200 μL suspension of coated spheroids in cell culture medium. Cells were incubated in the dark at 37 °C for 30 min. Stained cells were then visualised using confocal laser scanning microscopy (Olympus FV1000, Multiple Ar laser, Germany), and images were analysed using ImageJ software (NIH, USA).
The metabolic activities of coated and non-coated (control) MIN-6 cells were assessed using the Alamar Blue cell proliferation assay (Thermo Fisher, UK). The assay reagent reduces from a non-fluorescent blue colour to a fluorescent red form, during cellular metabolism. Hence, the amount of assay reagent reduction is proportional to cell number (presuming equal metabolic activity). Briefly, 20 μL of Alamar Blue reagent were added to 200 μL of cell spheroids suspension in DMEM, followed by incubating for 4 h at 37 °C. The fluorescence was then read (590 nm emission and 560 nm excitation) using a microplate reader (GloMax-Multi+, Promega, USA). The results were reported as percentage reduction between the coated and the control sample.