Bv421 mouse anti human cd235a

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.

Blood group H antigen triaose type I, blood group H antigen triaose type V (2’-fucosyllactose), blood group H antigen triaose type II, blood group A antigen triaose type V, blood group B antigen triaose type V were purchased from ELICITYL. 3-fucosyllactose was purchased from Carbosynth, GDP-Fucose, and Lewis a trisaccharide from Sigma Aldrich, and 6-fucosylchitobiose (GlcNAcβ(1-4)[Fucα(1-6)]GlcNAc) from TCI. Anti-blood group H ab antigen antibody [97-I] (ab24213; the epitope is a carbohydrate moiety) and mouse IgM [B11/7]-Isotype control (ab91545) were purchased from Abcam, anti-blood Group H n/ab antigen antibody [86-M] (AGM-022YJ) was purchased from Creative Biolabs, goat anti-mouse IgM (Heavy chain) cross-adsorbed secondary antibody PE (M31504) and CaptureSelect IgG-Fc were purchased from Thermo Fisher Scientific, and BV421 Mouse Anti-Human CD235a were purchased from BD Biosciences (cat. number 562938) Anti-H lectin reagent (extract of Ulex europaeus seeds) was purchased from BioRad and FITC-conjugated anti-H lectin (also an extract of Ulex Europeaus) (L32476) was purchase from Thermo Fisher Scientific. SialEXO, PNGase F, and FabRICTOR were from Genovis AB and Recombinant Fut2 was purchased from R&D systems. Fucosidase 95A from Bifidobacterium longum (CZ0511) was purchased from NZYTech.