Lsr fortessa sorp analyzer

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.

Samples were analyzed in 1 mL of cell suspension at a concentration of 1 × 10⁷ infected cells/mL in fresh complete medium without previous fixation or washing procedures, as previously described (66 (link)). Samples were analyzed on an LSR Fortessa SORP analyzer (BD Biosciences, San Jose, CA) equipped with a 488-nm blue laser for Vybrant DyeCycle green (1:500) (catalog number V350041; Thermo Fisher) detection (bandpass [BP] 530/30 nm) and a 405-nm violet laser for BV421 mouse anti-human CD235a antibody (1:500; BD-562938) and anti-GPA antibody (BP 450/50 nm) detection. Totals of 50,000 singlets were collected at a low flow rate to achieve the maximum resolution among subpopulations of iRBC and further analyzed with FCS Express Software version 7.12. Appropriate compensations were applied to all experiments. All parameters were processed using log scaling.