Celltiter blue viability assay

Cell Growth Curve was determined by taking transfected and control transfected cells and plating equal cell numbers into 12-well plate. Triplicate wells were counted for each transfection condition for each day, and the average was plotted on a graph. The variation in cell numbers is from multiple experiments not individual wells. Cell viability was measured using Cell Titer Blue Viability Assay obtained from Promega (G8080) and performed according to the manufacturer's instructions. Fluorescence was measured using a BioTek Synergy HT.

The Tocriscreen compound library (Tocris, Bristol, United Kingdom), consisting of 1,120 compounds (each stored as a 10 mM stock solution in DMSO), was added to confluent monolayers of HFFs in 96-well plates at a final concentration of 5 µM by automated pin transfer. Within 2 h after the addition of the drug, HFFs were infected with RH strain β-Gal–GFP-expressing (RH–β-Gal–GFP) parasites (from Gustavo Arrizabalaga, Indiana University) at an MOI of 1:5 (parasite/host cell ratio) and allowed to grow for 72 h before measurement of parasite growth with the β-Gal substrate CPRG (13 (link)). The number of parasites in each well was determined by linear extrapolation of standard curves generated in parallel in each plate. The effect of each compound on the viability of uninfected host cells was determined with the Cell Titer Blue viability assay (Promega, Madison, WI) after 72 h of growth. Average values and standard deviations of host cell growth were calculated, and those compounds that reduced host cell viability by 1.5 standard deviations were removed from further analysis. Follow-up assays with HFFs, MEFs, and MCF7 cells were performed under the same infection conditions.

To test in vitro drug sensitivity, tumor cells were plated in 96-well plates at optimal seeding densities in complete media and incubated overnight. Wells were then treated with varying doses of MK-4440 and/or IM. Cell proliferation and viability were measured at 72 h post treatment using the CellTiter Blue Viability Assay (Promega, Fitchburg, WI, USA) as described previously [24 (link)]. Assays were performed as three independent biological replicates, with a minimum of six technical replicates in each treatment arm. Combination indexes of CI_LD50 (GIST-T1) and CI_LD30 (GIST430, GIST-T1/829) were quantified using the previously described approach [24 (link)].

Cytotoxicity assays were performed in 96-well plates. GSCs were mechanically dissociated and plated at a density of 2.4×10⁴/ml, in triplicate for each treatment. Compounds were added 3 hours after seeding. Cell viability was estimated after 7 days by the chemiluminescence assay CellTiter-Glo™ (Promega Inc.) following manufacturer’s instructions. Vehicle control (DMSO) luminescence values were averaged and arbitrarily set to 100%. The absolute values of luminescence for each treatment were then normalized with respect to vehicle control and expressed as a percentage.
To evaluate doubling times, mechanically dissociated GSCs were plated in 96-well plates in triplicate and then incubated at 37°C in a 5% CO₂ incubator. Cell proliferation was monitored by counting cell number at different time points and confirmed by using the CellTiter-Blue Viability Assay (Promega Inc.).