Figure 6

Calcium handling in patients with mutated Gardos channel. (A) Representative confocal images of RBCs from controls and the patients loaded with the Ca²⁺-sensitive dye Fluo-4. A brighter fluorescence corresponds to a higher Ca²⁺-concentration. White arrows indicate sequestration of Ca²⁺ in intracellular vescicles. (B) Statistical analysis of wide field fluorescence Fluo-4 recordings for healthy donors (white bar, n = 2495 cells), patient II.4 (black shaded bar, n = 1257 cells) and patient III.1 (grey bar, n = 1402 cells). Because values are not Gaussian distributed we plotted boxes with whiskers from the 10^th to 90^th percentile. Significance was checked using the Mann-Whitney test; *, ***denote p < 0.05, and 0.001 respectively. (C–F) Intracellular Ca²⁺ (assessed as Ca²⁺-dependent fluorescence of Fluo-4) in RBCs of healthy controls and patients II.4 and III.1 measured by flow cytometry. Intracellular Ca²⁺ was measured at baseline and then the measurements were repeated immediately after the initiation of osmotic swelling by adding a bolus of distilled water to dilute the RBC suspension in isosmotic buffer by 1/3 (Swelling). (C) Readout for all RBCs in suspension; (D) Amount of cells forming “high Ca²⁺ fraction” (A-gated fraction, flow cytometric analysis shown in panel (E); (F) fluorescence intensity of Fluo-4 in this “high Ca²⁺ fraction”. All the experiments were performed in two occasions, and each time triple measurements were performed for each conditions. *, ** and ***denote p < 0.05, 0.01 and 0.001 respectively compared to healthy control. ^#, ^## and ^### stand for p < 0.05, 0.01 and 0.001 for osmotically compromised cells compared to the baseline values in either control or patients.