Calcein am

Cells were stimulated with 20 ng mL⁻¹ of the indicated cytokines for 12 h, then exposed to normoxic (20% O₂, 5% CO₂, 94%N) or hypoxic conditions (1% O₂, 5% CO₂, 94%N) for an additional 12 h, after which cells were stained with Calcein-AM (Invitrogen) following the manufacturer’s instructions. Briefly, wells were washed once with HBSS before adding a 2 µM Calcein-AM solution and Hoechst 33342 Solution (20 mM) in HBSS and incubated for 30 min at 37 °C. The total number of viable Calcein-AM positive cells were quantified by using ImageXpress Micro XLS Widefield High-Content Analysis System (Molecular Devices).
The release of lactate dehydrogenase by hypoxic cardiomyocytes was analyzed by using the CytoTox 96^® Non-Radioactive Cytotoxicity Assay (Promega) and performed according to the manufacturer’s instructions.

A co-culture model was conducted by culturing fibroblast cells and C. albicans together in a sterile 24-well plate, as adapted by Wong et al. (2014) (link), Oliveira Silva et al. (2018) (link). First, oral fibroblast cells were seeded in DMEM with 10% FBS at 37°C in 5% CO₂ for 24 h. The medium was then replaced with an inoculum of 5 × 10³ to 2.5 × 10³ CFU/mL C. albicans (MYA 2876) grown in DMEM without FBS. Fibroblast cells and C. albicans were treated with 33.28 μg/ml of A. colubrina extract. The plate was then incubated at 37°C in 5% CO₂ for 24 h. The vehicle control tested was 0.1% DMSO and the positive control was Fluconazole (10 μg/ml). The distribution of dead and live fibroblast cells was examined using the viability/cytotoxicity Live/Dead Assay Kit for mammalian cell type (Molecular Devices, Sunnyvale, CA), which contains a mixture of Calcein AM and EthDIII (Ethidium Homodimer III). Calcofluor white (Sigma Aldrich, San Luis, MO) was used to stain C. albicans. Fluorescent images of the double staining were captured using fluorescence microscopy (Keyence All-in-One BZ-X810 Fluorescence Microscope, Itasca, IL).