Fig. 1: Development of a flow cytometry osmotic stability assay.
From: Plasmodium vivax infection compromises reticulocyte stability
a Flow cytometry method for measuring RBC osmotic stability. Created with BioRender.com. b and c Representative flow cytometry forward scatter (FSC) side scatter by (SSC) plot (b) and FSC by FITC-phalloidin plot of a lysed (120 mOsm) RBC sample (c). Data representative of five independent experiments. d Representative immunofluorescent images of FITC–phalloidin binding RBC ghosts. Scale bar, 10 μm. Arrows indicate a RBC undergoing lysis. Data representative of two independent experiments. e Representative hemoglobin (Hgb) absorbance (black line) and flow cytometry (red line) lysis curves for normal RBCs. Error bars represent SD of n = 3 technical replicates. Data fit with least-squares regression fit curves of normalized data. f Normal (n = 6) and hereditary xerocytosis (HX) (n = 3) RBC lysis50 values measured by flow cytometry and hemoglobin absorbance lysis assays. Unique symbols indicate biological replicates and lines match the lysis50 values obtained from flow cytometry and hemoglobin absorbance assays. n.s. indicates no significant difference in lysis50 (normal RBC p = 0.8, HX RBC p = 0.1) using paired two-sided Student’s t-test. g Normal RBC (n = 12) lysis50 values measured by flow cytometry. Unique symbols indicate biological replicates. Horizontal lines and error bars represent mean ± SEM. h Normal RBC (n = 6) lysis50 values over the course of 14 days of 4 °C storage. Unique symbols indicate biological replicates. Data fit with linear regression lines. Mean linear regression for all data depicted by solid line. Pearson correlation of lysis50 and days of 4 °C storage, r = 0.15. i 4 °C degree stored (n = 3) and cryopreserved (n = 3) RBCs lysis50 values following transfer to P. vivax in vitro culture conditions. Unique symbols indicate biological replicates. Horizontal lines and error bars represent mean ± SEM. Asterisks denote significant differences in lysis50 during culture (4 °C degree stored RBCs p = 0.02, Cryopreserved RBCs p = 0.04) using RM one-way ANOVA analysis.
