Flow Cytometric Analysis of Erythrocytes

Twenty microliters of cell suspension was diluted in 80 μl of phosphate-buffered saline, stained with dihydroethidium (D7008; Sigma, Singapore) and SYBR green (10,000×; S9430; Sigma, Singapore) with 5 μg/ml and 5× as the final concentrations, respectively, and incubated for 20 min in the dark at room temperature as previously described (18 (link)). After staining, this suspension was analyzed using an Accuri C6 flow cytometer (BD Singapore). Forward scatter (FSC) and side scatter (SSC) signals of all erythrocytes were acquired via linear amplification, included potential abnormal cells due to hemoglobinopathies (22 (link), 23 (link)). Sixty thousand events were recorded, and flow cytometry analyses were done using FlowJo software (Tree Star). The proportion of schizont events for each sample was determined via a defined gating strategy (17 (link), 18 (link)).