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: Encysted cells were removed by scraping, pelleted, resuspended in PBS, and stained with 20 μM calcofluor white (Biotium) per manufacturer’s instructions.

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