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Figure 3 | Malaria Journal

Figure 3

From: A kinetic fluorescence assay reveals unusual features of Ca++ uptake in Plasmodium falciparum-infected erythrocytes

Figure 3

Effects of EGTA and SDS. (A, B) Addition of EGTA to chelate extracellular Ca++ 4 hours after initiating uptake (arrow in each panel; final [EGTA], 3.33 mM); the free Ca++ was buffered at 1.0 mM prior to EGTA addition. Panel A shows uninfected cells in the absence of probenecid. Here, EGTA addition abruptly reduces the fluorescence and prevents further increases (black circles); control kinetics are shown for resuspension of cells without addition of chelator (red circles). Panel B shows matched experiments for Dd2-infected cells in both the presence and absence of 5 mM probenecid (triangles and circles, respectively). With infected cells, the initial reduction with EGTA addition is smaller (black symbols), suggesting a lesser contribution of exported Fluo-8. EGTA prevents subsequent increases in fluorescence; probenecid-induced augmentation is also abolished (compare black triangles to black circles). These processes depend on extracellular Ca++ and uptake at the host membrane. (C, D) Addition of SDS to lyse cells 6 hours after initiating uptake with uninfected and Dd2-infected cells in the absence of probenecid (arrows; final [SDS], 0.05%). Note the large increases in fluorescence after addition of SDS (black circles), when compared to resuspension only kinetics (red or blue circles). (E) Effect of probenecid on plateau fluorescence after SDS addition, calculated by normalization of matched controls without probenecid to 100%. Bars represent mean ± S.E.M. of replicates from 2-3 independent experiments.

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