Bv421 anti mouse cd45

For the time course, mice were intravenously inoculated with 1 × 10⁶ mixed-stage iRBC. Blood and organs were harvested 2, 3, and 4 dpi as described above. Cells were processed for flow cytometry and stained with BV421 anti-mouse CD45 (BioLegend, catalog no. 103134), fluorescein isothiocyanate (FITC) anti-mouse CD71 (BioLegend, catalog no. 113806), phycoerythrin (PE)/Cy5 anti-mouse/human CD44 (BioLegend, catalog no. 103010), PE/Cy7 anti-mouse Ter119 (BioLegend, catalog no. 116222) at a 1:100 dilution, and eBioscience Fixable Viability Dye eFluor 506 (Invitrogen, catalog no. 65-0866-14) at a 1:1000 dilution.
For assessing parasite invasion, mice were intravenously inoculated with 4 × 10⁶ or 4 × 10⁷ purified schizonts. Blood and organs were harvested 1 hour after infection and processed for flow cytometry as described above. Cells were stained with Hoechst (Abcam, ab228551) at a 1:5000 dilution and BV510 anti-mouse CD45 (BioLegend, catalog no. 103137), FITC anti-mouse CD71 (BioLegend, catalog no. 113806), PE/Cy5 anti-mouse/human CD44 (BioLegend, catalog no. 103010), and PE/Cy7 anti-mouse Ter119 (BioLegend, catalog no. 116222) at a 1:100 dilution.

Flow cytometry was performed as previously described (Zhao et al., 2018 (link)). Briefly, kidneys were weighed and minced. A collagenase solution (1 mg/ml; Sigma-Aldrich, St. Louis, MO, United States) was used for renal digestion for 30 min at 37°C. A 100-μm cell strainer (Fisher Scientific) was used, together with a 1-ml syringe plunger, to acquire single cells. Cell pellets were washed and resuspended for further experiments. Anti-Mouse CD16/CD32 (553141; RRID:AB_394656, clone 2.4G2; BD Biosciences, San Jose, CA, United States) was used for non-specific Fc block. Antibodies used in this assay were acquired from BioLegend, San Diego, CA, United States; they include BV421 anti-mouse CD45 (RRID:AB_2562559), APC anti-mouse F4/80 antibody (RRID:AB_893481), PE anti-mouse CD206 (MMR) antibody (RRID:AB_10895754), FITC anti-mouse CD86 antibody (RRID:AB_313149), PerCP/Cyanine5.5 anti-mouse/human CD11b (RRID:AB_893232), APC/Cyanine7 anti-mouse CD3 antibody (RRID:AB_2242784), FITC anti-mouse CD4 antibody (RRID:AB_312713), and APC anti-mouse CD8a antibody (RRID:AB_312750). Data were acquired on a FACS Calibur cytometer [Becton Dickinson (BD), Bedford, MA, United States] and analyzed using FlowJo software (Tree Star, Ashland, OR, United States).

For FACS-sorting CD31 and PDGFRα populations, the following antibodies were used: FITC anti-mouse CD45 (Biolegend 103108), APC anti-mouse CD31 (Miltenyi 130-123-813), and PE-Vio770 CD140a (Miltenyi, 130-105-116). For analysis of endothelial cell death and mitochondrial ROS generation, the following antibodies were used: BV421 anti-mouse CD45 (Biolegend 103133), AF647 anti-mouse VE-Cadherin (Biolegend 138006). For these studies, mice were intravenously labeled with VE-Cadherin antibody (25 ug/mouse) via IV injection. Fifteen minutes following injection, mice were sacrificed, long bones were harvested and digested as described above. Endothelial cells were identified as the CD45- 7AAD- VE-Cadherin+ cell population. To discriminate apoptotic cells within this population, the CellEvent™ Caspase-3/7 Green Flow Cytometry Assay Kit (ThermoFisher, C10427) was used per manufacturer’s instructions. Mitochondrial ROS was measured using MitoSOX Red (ThermFisher, M36008).