Fig 1.
Graphical overview of the bioassay workflow.
After injection of the venom, chromatographic separation by HPLC is performed, followed by high-resolution fractionation on 96- or 384-well plates for subsequent cellular bioassaying and protein identification using proteomics as described by Slagboom et al. [27]. Image created using www.biorender.com.
Fig 2.
Digital phase contrast (DPC) and immunofluorescent microscopy images showing morphology of RPTEC/TERT1cells after 24 hours in presence of ten medically relevant snake species at increasing venom concentrations.
Both the DPC and fluorescent images were captured using confocal microscopy with 10X magnification. H342 staining is shown in blue, and PI in orange. All exposure settings were kept the same. The scale bar represents 200 μm. 0.1% Triton T-100 was used as a positive control. The images are scaled post-acquisition to the positive control. Yellow stars represent wells in which at least some activity was observed.
Fig 3.
Brightfield and immunofluorescent microscopy images showing the difference in morphology of RPTEC/TERT1 cells in presence of two venoms with contrasting effects.
(A) Cells exposed to 100 μg/mL E. ocellatus venom, with various channels (brightfield, Hoechst & PI) at 10X, 20X and 63X magnification. (B) Cells exposed to 100 μg/mL N. mossambica venom, with various channels (brightfield, Hoechst & PI) at 10X, 20X and 63X magnification. (C). Time series (0–150 min) of the effects of E. ocellatus venom (100 μg/mL), with a higher-magnification section (63X) clearly showing the monolayer detachment (60–70 min). (D). Time series (0–15 min) of the effects of N. mossambica venom (100 μg/mL) on cells, with a higher-magnification section (63X) showing the PI entering the cell (0–15 min). Scale bars lengths represented in the images.
Fig 4.
Comparison of morphological data and quantitative data for three venoms that have varying effects on the cells.
(A) Morphological data represented by immunofluorescent microscopy images showing morphology of RPTEC/TERT1cells after 24 hours exposure. Images were captured using confocal microscopy with 10X magnification. H342 staining is shown in blue, and PI in orange. 0.1% Triton T-100 was used as a positive control. Yellow stars represent wells in which activity was observed. Scale bar represents 200 μm. (B) Quantitative data of four cellular bioassays shown as bar graphs, with the activity of the venom represented relative to negative control (0 μg/mL). Live cell count (orange); cell surface area (grey); resazurin reduction activity (blue); ATP level (black). Increasing venom concentrations on the X-axes (in μg/mL) and percentage relative to negative control on the Y-axes. Measurements are presented as the mean of three individual experiments (N = 3), error bars depict SD; ‘*’ represents a statistically significant difference when compared to negative control, two-tailed test, p < 0.05 (Bonferroni-corrected).
Fig 5.
Identification of bioactive fractions of N. mossambica venom (1 mg/mL) by correlating bioactivity data with proteomics data.
i: bioactivity chromatograms obtained by plotting the results of three bioassays: cell surface area (grey); resazurin reduction (blue); live cell count (orange). The peaks with negative minima indicate the presence of bioactive fractions; ii: Graphs representing the protein score chromatograms (PSCs), showing the individual venom proteins found with Mascot database searching of the digested well fractions. iii: UV traces of the snake venoms at 220 nm obtained by RP-HPLC. The vertical outer lines mark the bioactivity window, which includes the main activity peaks and their corresponding PSC peaks and RP-HPLC-UV chromatogram peaks. Measurements are presented as the mean of three individual experiments (N = 3), error bars depict SD.
Fig 6.
Identification of bioactive fractions of E. ocellatus venom (5 mg/mL) by correlating bioactivity data with proteomics data.
i: bioactivity chromatograms obtained by plotting the results of two assays: resazurin reduction (blue); cell surface area (grey). The live cell count was not included as the graphs do not accurately describe the number of live cells as a result of the detachment of the monolayer (see also above). The peaks with negative minima indicate the presence of bioactive fractions; ii: Graphs representing the protein score chromatograms (PSCs), showing the individual venom proteins found with Mascot database searching of the digested well fractions; iii: UV traces of the snake venoms at 220 nm obtained by SEC-HPLC. The vertical outer lines mark the bioactivity window, which includes the main activity peaks and their corresponding PSC peaks and SEC-UV chromatogram peaks. Measurements are presented as the mean of three individual experiments (N = 3), error bars depict SD.