Figure 3. Detection of cytosolic Ca²⁺ levels during merozoite invasion.

(a–d) Cytosolic Ca²⁺ levels were detected using a fluorescence plate reader. (a) Preloaded T994 merozoites with Fluo-4AM were incubated with erythrocytes in the absence (RBC) or presence of Cyto D (RBC+Cyto D). (b,c) Preloaded T994 (b) or T994ΔRH1 (c) merozoites were incubated with erythrocytes in the absence (RBC) and presence of either C41 (RBC+C41 (0.2 mg ml⁻¹)) or C2 (RBC+C2 (0.2 mg ml⁻¹)). (d) Preloaded T994 merozoites were incubated with erythrocytes in the absence (RBC) or presence of mAb C41 Fab fragment (C41 Fab (0.2 mg ml⁻¹)). The cumulative changes of Ca²⁺ levels (≤ΔF (t)) in merozoites over 600 s were summed up and plotted against time (s). Experimental data were presented as the mean±s.e.m.; n=3. *P≤0.0001 by one-way ANOVA, indicating that C41 successfully blocked the Ca²⁺ signalling in T994 when compared with the absence of C41. ^#P≤0.0001 by one-way ANOVA, indicating that C41 successfully blocked the Ca²⁺ signalling in T994 when compared with the presence of C2.⁺P≤0.0001 by one-way ANOVA, indicating that C41 Fab fragment significantly inhibited the Ca²⁺ signalling in T994, when compared with the absence of C41 Fab fragment. (e–g) Cytosolic Ca²⁺ levels detected by flow cytometry. Representative FACS contour plots (e,f) were gated by size (FSC-A) versus mean fluorescence (MF) of Fluo-4 AM of merozoite (DAPI (+)) incubated with erythrocytes in the absence (e, Positive) or presence of C41 (f, C41). Vertical gating is to separate the free merozoites with low FSC from the RBC-attached merozoites with high FSC, whereas horizontal gating is to differentiate intercellular Ca²⁺ levels. Q1 (green) represents free merozoites with Fluo-4 AM signals. Q2 (green) represents invading merozoites with increasing Fluo-4 AM signals. The number (blue) in Q2 represents % population of attached merozoites. Q3 (peach) represents free merozoites with no signal. Q4 (pink) represents attached merozoites with no signal. (g) Statistical analysis of the effect of C41 on Ca²⁺ signalling during T994 merozoite invasion by FACS. C41 was able to reduce the number of invading merozoites to 56.6% (blue) and to decrease the overall Ca²⁺ level to 32.42% (red) during invasion, as compared with that in the absence of C41. Experimental data are presented as the mean±s.e.m.; n=3.