Vybrant dyecycle green

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.

Cell cycle distribution was evaluated 24 h after cell treatment with the respective IC₅₀ of GSH-NS B or GSH-NS D. The occurrence of the so-called sub-G₀/G₁ peak, which is a distinct cell population characterized by subdiploid DNA fluorescence and might correlate with the internucleosomal DNA fragmentation typical of apoptosis (Pozarowski and Darzynkiewicz, 2004), was also evaluated. Briefly, 1 × 10⁶ HCT116, 1 × 10⁶ HT-29, 1 × 10⁶ DU145, and 1 × 10⁶ PC-3 cells were incubated with 2 µM of the live cell staining Vybrant Dye Cycle Green (Invitrogen) for 30 min at 37 °C. The samples were run on a flow cytometer with 488 nm excitation to measure Vybrant Dye Cycle Green staining and data analysis was performed by FCS Express software version 4 (BD Bioscience, Milano, Italy).