Cell freezing medium

Fresh human bone marrow aspirate was purchased from Lonza (donor 18‐year‐old black female) and the hMSCs were isolated based on preferential adhesion to TCPS plates, using previous published protocols.20, 28 Freshly isolated hMSCs (P0) were detached with 0.05% trypsin–EDTA (Sigma) and subsequently centrifuged, counted, and frozen down in 80% fetal bovine serum (FBS; Invitrogen) and 20% dimethylsulphoxide and stored in liquid nitrogen. For passaging, hMSCs were cultured for 3 days on TCPS at an initial density of 4,000 cells/cm² in expansion media, detached with 0.05% trypsin‐EDTA, centrifuged, and replated at the same density. Expansion media consisted of low glucose (1 ng/mL glucose) Dulbecco's Modified Eagle Medium (ThermoFisher) supplemented with 10% FBS (ThermoFisher), 1 ng/ml fibroblast growth factor basic (Life Technologies), 50 U/ml penicillin (ThermoFisher), 50 μg/ml streptomycin (ThermoFisher), 0.5μg/ml of Amphotericin B (ThermoFisher). This method was repeated to generate desired passage numbers. For subsequent analyses, cells at desired passage numbers (P2 for early, P5–P7 for middle, and P11–P12 for late) were frozen in cell freezing medium (Sigma) and stored in liquid nitrogen.

Blood samples were processed for isolation of Ehrlichia in cell culture. To this end, the blood was aseptically collected in EDTA vacuum tubes and transported to the laboratory for mononuclear cells (MNCs) isolation, using Histopaque 1083 (Sigma-Aldrich, St. Luis, MO, USA), as previously described [19 (link)]. Cultures were initiated by seeding the MNCs in a 25 cm² culture flask containing Dulbecco’s Modified Eagle’s medium (DMEM, Sigma-Aldrich, St Louis, MO, USA) and supplemented with 20% iron-fortified Bovine Calf Serum (BCS, Sigma-Aldrich, St Louis, MO, USA). The culture flask was kept at 37 °C and 5% CO₂. Every two days, 1/5 of the primary culture medium was collected for cytologic evaluation, using Romanowsky-stained smears (NewProv, Pinhais, PR, Brazil), and fresh medium was added. After 96 hours (h) of incubation, DH82 cells were added to the primary culture of MNCs, and the supplementation of DMEM was reduced to 5% iron-fortified BCS. The culture was kept at 37 °C and 5% CO₂. Every seven days, samples were collected for cytologic evaluation and PCR. Stocks of infected DH82 cells were resuspended in cell-freezing medium (Sigma-Aldrich, St Louis, MO, USA) and frozen at −156 °C in liquid nitrogen.

In all experiments, sacrifices were carried out in the morning (from 9 am to 1 pm). Mice were anesthetized, exsanguinated by heart puncture, and transcardially perfused with PBS. For plasma metabolite analysis, blood was collected in tubes containing 20 μL heparin and spun at 3,000 g for 15 min at 4 °C. Supernatant plasma was aliquoted and snap frozen in liquid nitrogen (LN). For immune profiling, peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation protocol (Cytiva™ – density 1.084 ± 0.001 g/ml). After dissection, the following brain regions were excised: for histology, left hemisphere, post-fixed in 4% paraformaldehyde (PFA)/PBS; for sNuc-Seq, left hippocampus, snap frozen in LN; for ELISA and biochemistry, right cortex and right hippocampus, snap frozen in LN. The spleen was mashed with the plunger of a syringe against a 70 mm strainer and treated with ammonium-chloride-potassium (ACK) lysis buffer (Gibco™) to remove erythrocytes. Splenocytes were then used immediately for flow cytometry and pan-T-cell cultures, while for CyTOF, aliquots were resuspended in cell freezing medium (Sigma-Aldrich) and frozen in a Mr. Frosty container (Thermo Fisher) at −80 °C. The left gonadal fat pad was snap frozen in LN for sNuc-Seq.