Apc rat anti mouse ter 119

Parasitemia measurements for B. divergens and B. microti were done using previously established protocols in our lab (Cursino-Santos et al., 2017 (link)). Briefly, mouse erythrocytes (1 × 107 cells/ml) were identified by allophycocyanin (APC) rat anti-mouse TER-119 at a final concentration 0.005 μM (BD Pharmingen). iRBCs were identified by staining parasite DNA using Hoescht 33342 (0.1 μM final concentration; Thermo Fisher Scientific). As all RBCs lack a nucleus, RBCs with a positive signal for DNA represent infected host cells bearing parasites. For in vitro cultures of B. divergens in human blood, samples were stained with the DNA-dye Vybrant® DyeCycle™ Green (1:500) and BV421 mouse anti-human CD235a (BD-562938; 1:500), which labels human RBCs. Samples were analyzed on an LSR Fortessa SORP analyzer (BD Biosciences), equipped with a 355-nm UV laser for Hoechst detection (361/486 nm), a 640-nm red laser for APC–TER-119 detection [650/60 nm bandpass (BP)], a 488-nm blue laser for Vybrant® DyeCycle™ Green detection (530/30 nm BP), and a 405-nm violet laser for anti-GPA detection (450/50 nm BP) in 10,000 target events (iRBCs). The forward scatter threshold was set on 300, and 10,000 total events were acquired at “low” flow rate. FACSDiva software (version 6.2; BD Biosciences) was used for data analysis. All parameters were processed using log scaling.

Expression of red cell surface markers was analyzed on a FACS Calibur instrument (Becton Dickinson, Lincoln Park, NJ, USA). Cells were washed and re-suspended in PBS containing 0.1% BSA (FACS buffer), and stained with fluorescence-labeled antibodies. Data analyses were performed with FlowJ software (TreeStar). Flow cytometry antibodies were purchased from BD Biosciences: APC rat anti-mouse Ter119 (cat^# 557909) and PE or FITC labeled rat anti-mouse CD71 (cat^# 553267 or 553266, respectively, San Jose, CA, USA). Erythroid subpopulations (nucleated erythroblasts and reticulocytes) were sorted using a FACSAria cytometer (BD Biosciences) based on CD71/Ter119 expressions. CD71⁺/Ter119⁺/FSC^high cells were defined as nucleated cells, whereas CD71⁺/Ter119⁺/FSC^low cells were considered reticulocytes [20 (link)]. To measure hydrogen peroxide (H2O2)-induced red cell lyses, whole blood diluted at 1:500 in PBS was incubated with different concentrations of H2O2 at room temperature for 5 minutes, shook vigorously for 15 seconds, and analyzed immediately on forward scatter channel (FSC) and side scatter channel (SSC) using flow cytometry.