Viability/Cytotoxicity Assay Kit for Animal Live & Dead Cells

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Prepare a staining solution of 2 uM calcein AM/4 uM EthD-III by adding 5 uL of 4 mM calcein AM and 20 uL of 2 mM EthD-III to 10 mL of PBS or other serum-free buffer or medium	Incubate the cells for 30-45 minutes at room temperature.

Upstream tips
Prepare a staining solution of 2 uM calcein AM/4 uM EthD-III by adding 5 uL of 4 mM calcein AM and 20 uL of 2 mM EthD-III to 10 mL of PBS or other serum-free buffer or medium
Protocol tips
Incubate the cells for 30-45 minutes at room temperature.

Publication protocol

A co-culture model was conducted by culturing fibroblast cells and C. albicans together in a sterile 24-well plate, as adapted by [32]. First, oral fibroblast cells (ATCC: CRL2014) were seeded in Dulbecco’s Modified Eagle’s Medium (DMEM) with Fetal Bovine Serum (FBS) (Gibco) at 37°C in 5% CO2 for 24 hours. The medium was then replaced with an inoculum of 5x103 to 2.5x103 CFU/ml C. albicans (MYA2876) grown in DMEM without FBS. Fibroblast cells and C. albicans were treated with 62.5 μM of lichochalcone-A. The plate was then incubated at 37°C in 5% CO2 for 24 hours. The vehicle control tested was 1% ethanol and the positive control was fluconazole (32 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).

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