Figure 2: CellTiterGlo can be used to assay Caco-2 cell killing by amoebae.

Caco-2 cells were cultured and harvested as described [7]. 18–24 hours prior to performing the experiment, Caco-2 cells were plated in 96-well plates. On the day of the experiment, cells were washed with fresh TYI. Amoebae were added to create approximate co-incubation ratio of 1 amoeba: 10 Caco-2 cells. The CellTiterGlo assay (Promega) was carried out according to the manufacturer’s instructions. Three wells of amoebae alone, six wells of Caco-2 cells alone, and six wells of co-incubated samples were averaged to obtain one value per condition, and three experiments were performed independently on different days. (A) A dilution series of amoebae, or (B) human Caco-2 intestinal epithelial cells were assayed using CellTiterGlo. Best fit lines and R² values are shown. CellTiterGlo signal correlates with the number of cells per well. Data represent the average values of two replicate wells for each cell concentration, and are representative of 3 independent experiments. (C) Amoebae were co-incubated (filled circles) with Caco-2 cells at an approximately 1:10 ratio, or amoebae (filled triangles) and Caco-2 cells (filled squares) were incubated separately as controls. Data represent the average values of two replicate wells for each sample, from one experiment. (D) Data from 3 independent experiments performed as in panel C were normalized to the value of each sample at Time = 0. There were statistically significant differences between the co-incubation and Caco-2 alone samples, as indicated. GraphPad Prism was for student’s unpaired t test statistical analysis. Mean values and standard deviations are shown; ns = P > 0.05, * = P < 0.05, ** = P < 0.01, *** = P < 0.001, **** = P < 0.0001.