Journal: bioRxiv

Article Title: A high-throughput, image-based assay to assess drug sensitivity of Acanthamoeba castellanii cysts

doi: 10.1101/2025.06.06.658337

Figure Lengend Snippet: A: Conditions from figure S4 with a mean A565 ≥ 2.5. Components of each formulation listed in Table S2. B: Trophozoites were encysted in the same 4 conditions shown in A, but with 24 replicate wells per condition to assess for variability in plate location. C: Microscopy of cells from B at end of encystment. Phase images acquired using an EVOS FL Auto Imaging System (20x objective). D: Cells were encysted in FEM (ingredients listed in inset; bolded items are new components unique to FEM, unbolded items are ingredients in EMb) for 48 (blue) or 72 (red) hours. E: Cells encysted in FEM were stained with 20 µM calcofluor white and imaged using a Nikon Eclipse Ti-E inverted microscope (100x objective with oil) in a glass-bottom dish overlaid with a 1.5% agarose pad. Representative images displayed. Arrow, immature cyst. F: Cells were encysted using our former protocol in EMb for 48 hours (blue) or our optimized method in FEM for 72 hours (red). A, B, D, F: Scatter plots report the absorbance measured by SRB assay after SDS treatment. The mean and SEM of at least 3 independent experiments with at least 3 replicate wells per condition are shown. Statistical differences between 3 or more groups calculated with one-way ANOVA (Brown-Forsythe and Welch ANOVA tests for unequal SDs) with Dunnett’s T3 multiple comparisons test (A) or with Games-Howell multiple comparisons test (B, for n>50); differences between 2 groups calculated with two-tailed, unpaired t test with Welch’s correction (F); ns for not significant (p>0.05), **** for p<0.0001.

Article Snippet: Phase-brightfield and fluorescent (using GFP and TRITC channels to image c-AM and EthD-1 staining, respectively) images of wells were then taken with an automated Nikon Eclipse Ti-E inverted microscope (20x objective) and an Andor Zyla VSC-01400 camera with 3 separate Z stacks taken with 5 μm steps per channel, per well.

Techniques: Formulation, Microscopy, Imaging, Staining, Inverted Microscopy, Sulforhodamine B Assay, Two Tailed Test

Journal: bioRxiv

Article Title: A high-throughput, image-based assay to assess drug sensitivity of Acanthamoeba castellanii cysts

doi: 10.1101/2025.06.06.658337

Figure Lengend Snippet: A: The manual cell counts (x axis) reported in figure S6 were compared to those generated using the automated image analysis pipeline (y axis). Dashed line represents the perfect fit line y = x. B: Quantification of cysts described in figure S5. Live cysts in FEM and those that were heat-killed (HK) by autoclaving were incubated with fluorescent live/dead stain (blue) or PBS control (red), imaged with a Nikon Eclipse Ti-E inverted microscope (20x objective), and counted using automated image analysis pipeline. The mean and SEM of at least 3 independent experiments with at least 3 replicates per condition are shown. % red, percent of total cells stained with EthD-1; % green, percent of total cells stained with c-AM; % red is green, percent of red cells that also stained with c-AM. C, D: Cysts were treated with 0.5% bleach, 10% H 2 O 2 , 20 µM chlorhexidine, or media controls for 20 hours and compared to live cysts in FEM, heat-killed (HK) cysts, or a 1:1 mix of HK and live cysts in FEM (mix). Cells were imaged and stained with the fluorescent live/dead staining assay and counted using automated image analysis pipeline. Chlorhexidine was dissolved in DMSO; bleach and H 2 O 2 were dissolved in PBS; all three were added at 1% final volume in PBS. C: Percent survival calculated by subtracting the percent of EthD-1-stained cells (measured by fluorescent live/dead staining) from 100%. The mean and SEM of at least 3 independent experiments with at least 3 replicates per condition are shown. Statistical differences calculated with one-way ANOVA (Brown-Forsythe and Welch ANOVA tests for unequal SDs) with Dunnett’s T3 multiple comparisons test. D: Microscopy of cysts following treatment. Images acquired using a Nikon Eclipse Ti-E inverted microscope (20x objective). Representative images displayed.

Article Snippet: Phase-brightfield and fluorescent (using GFP and TRITC channels to image c-AM and EthD-1 staining, respectively) images of wells were then taken with an automated Nikon Eclipse Ti-E inverted microscope (20x objective) and an Andor Zyla VSC-01400 camera with 3 separate Z stacks taken with 5 μm steps per channel, per well.

Techniques: Generated, Incubation, Staining, Control, Inverted Microscopy, Microscopy