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Figure 3 | Malaria Journal

Figure 3

From: Cytoplasmic free Ca2+ is essential for multiple steps in malaria parasite egress from infected erythrocytes

Figure 3

Manipulating the free calcium concentration in schizonts affects parasite egress. A. Reducing the extracellular free Ca2+ concentration does not affect parasite egress. Synchronized cell cultures approaching the end of the cycle were pretreated with different concentrations of EDTA, pH 7.4, for 5 min at 37°C to chelate external Ca2+ and then observed. Drug-pretreated and control cultures were kept for two hours at 37°C to accumulate sites of egress and then egress was evaluated as described in the Methods and presented as Mean ± SEM, n = 3-4 independent experiments; 7,429 infected cells were analyzed. Concentration of free calcium was less than 10 nM according to a fluorimetric assay for free Ca2+. B. Reduction in intracellular Ca2+ concentration by BAPTA inhibits parasite egress. Synchronized cell cultures approaching the end of the cycle were pretreated outside the chambers with increasing concentrations of the cell permeant Ca2+ chelator BAPTA AM at 37°C for 30 min. Pretreated cultures were then injected into the chambers for microscopy and kept at 37°C for 90 min to accumulate sites of egress and then parasite egress was assessed in treated and control cultures as described above (mean ± SEM, n = 3-4 independent experiments; 9,669 infected cells were analyzed). C. The Ca2+ ionophore A23187 activates parasite egress only in the presence of intra-and extra-cellular Ca2+ and upon short (15–30 min) treatment of infected cells. Cells pretreated with EDTA (5 min at 37°C) or BAPTA AM (30 min at 37°C) or both were injected into the chambers immediately after addition of ionophore into the medium. Chambers were kept at 37°C for 15–30 min to accumulate sites of egress and then parasite egress was assessed in cultures as described above (mean ± SEM, n = 2-6 independent experiments, at least 300 infected cells were analyzed for each condition in each experiment).

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