Celltiter glo luminescent cell assay

The amount of intracellular ATP produced by mycoplasma-free and mycoplasma-infected T. vaginalis G3 was evaluated in three different phases of growth: lag, exponential, and stationary phase. Furthermore, the amount of ATP produced by microorganisms grown in media additioned with 0.1 mM or 0.5 mM L-Arginine was also compared. A total of 15,000 cells of the parasites in exponential growth phase for each tested condition was harvested and centrifuged at 4000 × g for 10 min and the amount of ATP analyzed in 50 μl by using CellTiter-Glo Luminescent Cell Assay (Promega) according to the manufacturer's instructions.

SNU-5 and EBC-1 cancer cells were seeded in 96-well plates (2000 cells/well) in the appropriate culture medium in the presence of 10% fetal bovine serum (FBS). After 24 h, cells were treated with increasing concentrations (0–5 μM) of long-sc60, short-sc25, or MvDN30 in fresh culture medium supplemented with 5% FBS. After 72 h of treatment, cell number was determined using CellTiter-Glo Luminescent Cell Assay (Promega, Madison, WI, USA) with a VICTOR X4 multilabel plate reader (Perkin Elmer Inc.).

MTS assay (Molecular Probes, ThermoFisher Scientific, Rockford, IL, USA) or the CellTiter Glo Luminescent Cell Assay (Promega, Madison, WI, USA) were used to establish proliferation and viability of tumor cells, under previously reported conditions.^{15 (link), 22 (link)} Apoptosis was measured by annexin-V and propidium iodide staining of cells followed by flow cytometry analysis. The conditions and method for these assays have been previously described by us.^{15 (link)} Drug combination synergy, additivity or antagonism were determined using the CompuSyn Software (ComboSyn Inc., Paramus, NJ, USA), which uses the Chou-Talalay principle.^{23 (link)} Details for this analysis are presented in the Supplementary Material and methods.