Viability cytotoxicity assay kit for animal live dead cells

Cytotoxicity assays using fluorometric quantification of cellular viability were performed on oral fibroblast cells (ATCC: CRL2014). Fibroblast cells (1 × 10⁵ cells/ml) were first seeded in a 96-well plate in DMEM medium with 10% FBS and incubated at 37°C in 5% CO₂ for 24 h. Cells were then treated with 1-monolaurin (3.9–2000 µM) and incubated for an additional 24 h. Cell viability and morphological characteristics were observed by fluorometric method and microscope, respectively (National Committee for Clinical Laboratory Standards (NCCLS) , 2002 ; O’Brien et al. (link), 2000 (link); Pasetto, Pardi & Murata (link), 2014 (link)). The medium was then replaced with an inoculum of 5 × 10³ to 2.5 × 10³ CFU/ml C. albicans (ATCC: SC5314) grown in DMEM without FBS. Fibroblast cells and C. albicans were treated with 125 µM and 250 µM of 1-monolaurin. The plate was then incubated for 24 h. The vehicle control tested was 1% ethanol while the positive control used was fluconazole (32.2 µM). The distribution of dead and live fibroblast cells was examined using the Viability/Cytotoxicity Assay Kit for Animal Live & Dead Cells (Biotium), which contains a mixture of Calcein AM and EthDIII. Calcofluor white (Sigma) was used to stain C. albicans. Fluorescent images of the double staining were captured using fluorescence microscopy (EVOS fl microscope AMG, Bothell, WA, USA).

Live & Dead cell assay was performed using viability/cytotoxicity assay kit for Animal Live & Dead Cells (Biotium, Fremont, CA, USA). According to manufacturer’s protocol, live-dead cell study was performed on treated MCF-7 and MDAMB-231 spheroids on day 10th of both single and multiple dosing in the therapeutic model. Briefly, after complete removal of media from the wells; 100 μL of staining solution (2 μM calcein AM/4 μM Ethidium homodimer III (EthD-III)) was added to treated spheroids; in order to give the green/red fluorescent staining for viable and dead cells, respectively. Plate was then incubated at room temperature for 45 min in dark. Images were captured at 4× magnification using fluorescence microscope (EVOS-FL, Thermo Fisher Scientific, Waltham, MA, USA).