Cell staining, flow cytometry and fluorescence-activated cell sorting (FACS)

Parasite cultures were analysed on a MACSQuant Analyzer 10 (Miltenyi Biotec) after a 10 min centrifugation at 4,105×g and resuspension in PBS + 0.2% BSA buffer. Analyses were performed using FlowLogic Software (Miltenyi Biotec) using a specific gating for singlet parasites expressing dsRed, for which the non-transfected parental parasite line served as a control. In some experiments, parasites were stained with 5-(and 6)-carboxyfluorescein diacetate succinimidyl ester (CFSE; Cell Division Tracker Kit, BioLegend) according to manufacturer’s instructions. Briefly, lyophilized CFSE was reconstituted with DMSO to a stock concentration of 5 mM. This stock solution was diluted in PBS to a 5 μM working solution. Promastigotes at a concentration of 10⁸ cells/mL were centrifuged at 4,000×g for 10 min and resuspended in CFSE working solution for 20 min at 25°C. The staining was quenched by adding 5 times the original staining volume of cell culture medium containing 10% FBS. Parasites were centrifuged again and resuspended in pre-warmed HOMEM medium for 10 min. After incubation, CFSE labeled parasites were used for infection and determining in situ proliferation in macrophages, LT-HSC and human HSPC.

Sorting of mouse LT-HSC and human HSPC was performed, and quality confirmed as described previously [16]. Briefly, BM cell suspensions (2×10⁷/mL concentration) were treated with FcɣR-blocking agent (anti-CD16/32, clone 2.4G2, BD Biosciences) for 15 min, followed by a washing step using 400×g centrifugation and resuspension in PBS + 0.2% BSA buffer. Next, cells were incubated for 20 min at 4°C with a mix of fluorescent conjugated anti-mouse antibodies at optimized concentrations. DAPI Staining Solution (Miltenyi Biotec) was used to assess viability. A 96-well plate (Greiner Bio-One) was prepared for sorting by adding RPMI 1640 medium supplemented with 1% NEAA, 100 U/mL penicillin, 100 μL streptomycin, 500 μg/mL gentamycin, 2 mM L-glutamine, 1 mM sodium pyruvate and 10% iFCS to the wells in which 10,000 LT-HSC/well were sorted using FACSMelody (BD Bioscience) following specific gating strategies, confirmed with fluorescence minus one (FMO) controls and compensated using single stains, as described and shown in S7 Fig and Tables 1–3 of our previous report [16]. For visualizing infection, LT-HSC were collected on slides by Cytospin, fixed using methanol and stained for 15 min with Giemsa (Sigma Aldrich). Microscopic images were acquired using the UltraVIEW VoX dual spinning disk confocal system (PerkinElmer). For image analysis, z-stack imaging was included and a z-projection using the max intensity method was created for each image. The region-of-interest (ROI) was then manually drawn around the DsRed⁺ signals of the amastigotes, allowing quantification of the area and fluorescence intensity using the FIJI software. For analysis of dsRed and/or CFSE levels on amastigotes at designated timepoints, infected macrophages and LT-HSC were recovered from the cultures. Cells were centrifuged at 400×g for 10 min and in PBS + 0.2% BSA. Host cell membranes were disrupted by 3 passages through a 25G needle. Amastigotes were collected in the supernatant after centrifugation at 250×g for 10 min and subsequently pelleted at 3,000×g for 10 min and resuspended in 500 μL PBS + 0.2% BSA for analysis by flow cytometry.