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. 2024 Jan 3;16(1):2297451. doi: 10.1080/19420862.2023.2297451

Figure 3.

Figure 3.

Real-time complement dependent cytotoxicity of hF5-WT and hF5-LALA. Schematic diagram illustrating RT-CDC assay conditions (a). Percent cell viability of Vero cells over the course of VEEV-TC83 infection for 48 h with hF5-WT and hF5-LALA, wells spiked with mouse (b) or human (c) sera at 8hpi (red arrows). % viability = [(number of blue cells – number of red cells)/number of blue cells] *100. Blue signal indicates Hoechst+, red and blue indicates Hoechst+ propidium iodide+. Images of NMS and NHS assays are taken from timecourse videos that span 9-25hpi (hours post infection). All image snapshots are taken at 24hpi. NMS treated hF5-WT (D, S1 video), hIgg1 (E, S2 video), and hF5-LALA (F, S3 video). NHS treated hF5-WT (G, S4 video), hIgg1 (H, S5 video), and hF5-LALA (I, S6 video). CDC activity was pronounced in hF5-WT treated wells with both mouse and human sera, whereas hF5-LALA mutant treatment with mouse or human sera showed significantly reduced cytotoxicity. In CDC-NMS assay, hIgg1 v hF5-WT p < 0.0001, hF5-WT v hF5-LALA p < 0.0001, hIgg1 v hF5-LALA p = 0.0001. In CDC-NHS assay, hIgg1 v hF5-WT and hF5-LALA p < 0.0001, hF5-WT v hF5-LALA p = 0.0013, by paired t-test. Data shown is representative of three or more experiments, each with three technical replicates (b and c), quantified using CellInsight C×7High Content Analysis software. Images (d-i) taken at 10X magnification on a CellInsight C×7High Content imaging platform.