Cd45.2 fitc

Specific-pathogen free, 6-10 week old, male and female B6.Cg-Tg(K18-ACE2)2Prlmn/J (K18-hACE2; stock #034860) mice were purchased from The Jackson Laboratory and/or bred in-house. For SARS-CoV-2 infections, mice were intranasally inoculated with 10¹-10⁴ PFU in 25 μl PBS to a single nare after anesthetizing with ketamine/xylazine. Prior to euthanizing animals for tissue harvest, mice were anesthetized with ketamine/xylazine and then retro-orbitally injected with 2.5 μg CD45.2 FITC (Biolegend) in 100 μl PBS to label circulating cells. All animal studies were approved by and conducted in accordance with Rocky Mountain Laboratories’ Animal Care and Use Committee under animal study protocol 2020-075-E.

Cells were suspended in Ca⁺⁺ Mg⁺⁺-free phosphate-buffered saline (PBS) containing 2% (vol/vol) FBS (Globalstem). Human hematopoietic progenitors cells were identified by labeling with CD45-PE (1:50; clone HI30) and or CD34-APC (1:50; clone 561) while mature phenotypes were analyzed using CD15-Pacific Blue (monocytes and granulocytes) (1:50; clone W6D3/HI98), CD14-FITC (monocytes and macrophages)(clone 1:50; M5E2); CD11b (1:50; ICRF44) (macrophages) and Glycophorin-PECy5 (red blood cell membrane), and erythroid precursors (1:50; Clone HIR2) (all from Biolegend). Murine hematopoietic cells were identified using the following antibodies: Mac-1/CD11b-PECy7 (1:200; clone M1/70), Gr-1-PE (1:200; clone 8C5), B220-PECy5 (1:100; clone RA3-6B2), Sca1-APC (1:100; clone E13-161-7), c-Kit-APCCy7 (1:100; clone 2B8): CD45.1-APC (1:100; clone A20) and CD45.2-FITC (1:100; clone 104) (all from Biolegends). All antibodies were incubated at the concentration suggested by the manufacturer for 30 minutes on ice. Non-specific signals and dead cells were excluded by appropriate fluorochrome-conjugated isotype and propidium iodide staining, respectively. Cell fluorescence was analyzed with the FACSAria II (Becton Dickinson).

Plasmodium yoelii (clone 17XNL, obtained from MR4, ATCC) and Plasmodium chabaudi chabaudi AS(Pyr1) (clone MRA-747, obtained from beiresources) infections were initiated by a serial transfer of 10⁶ parasitized red blood cells (pRBCs) i.v. Control RBCs were isolated from naive C57BL/6 mice aged 6–8 weeks, 10⁶ RBCs were transferred. To inactive iRBCs, 5mLof iRBCs were irradiated with 200 Gy by cesium irradiation. Parasitemia was measured using flow cytometry as previously described (Malleret et al., 2011 (link)). Briefly, 1 mL of blood was obtained by milking the tail vain and mixed to 100uL of PBS. The cells were spun down and resuspended in 100 mL of staining cocktail containing Hoechst-34580 (1:1000, Thermo Fisher Scientific), Dihydroethidium (1:500, Thermo Fisher Scientific), CD45.2-FITC (1:200, clone:104, Biolegend) and TER-119-APC (1:400, clone: TER-119, Tonbo) and incubated for 30 minutes at 4°C. The cells were washed with PBS and spun down at 200 x g for 5 minutes and run on FACSVerse after resuspending in 100mL of PBS. Percentage RBCs staining positive for Hoechst 34580 and Dihydroethidium indicative of parasitized cells were calculated using FlowJo software (Treestar Inc.).