Methylene blue

The hippocampus, SVZ, anterior and posterior cerebrum, thalamus, cerebellum, brain stem and spinal cord from the fetal CNS between 14–23 weeks of gestation (n = 11) were isolated and mechanically minced with a scalpel, enzymatically dissociated in 0.25% trypsin for 15 min at 37°C, which was quenched with an equal volume of 40 mg/ml of BSA suspended in Earles' balanced salt solution. The resulting cell suspension was then filtered through 70 µm filters (BD Biosciences, Franklin Lakes, NJ) and washed twice with PBS before enumeration. Viability of cells was determined with 3% acetic acid with methylene blue (StemCell Tech, Canada).

PB of mice was collected retro-orbitally, and hematopoietic parameters were measured by complete blood counts. Central bone marrow was flushed from femurs, and cellularity was quantified with 3% acetic acid in methylene blue (STEMCELL Technologies, 07060). For flow cytometric analysis of the lineage cell compartment of mice, flushed bone marrow cells were stained for myeloid (rat anti–Gr-1 APC-Cy7, BD Biosciences, 557661; rat anti–Mac-1 APC, eBioscience, 17-0112-83) or lymphoid lineages (rat anti-B220 FITC, eBioscience, 11-0452-82; hamster anti-CD3 PE-Cy7, eBioscience, 25-0031-82). The HSPC compartment was analyzed by staining for Lineage (biotin–Ter-119, –Mac-1, –Gr-1, -CD4, -CD8α, -CD5, -CD19, and -B220, eBioscience, 13-5921-85, 13-0051-85, 13-5931-86, 13-0112-86, 13-0452-86, 13-0041-86, 13-0081-86, 13-0193-86) followed by staining with streptavidin–PE-Texas Red (Invitrogen, SA1017), rat anti-cKit APC-Cy7 (eBioscience, 47-1171-82), rat anti-Sca1 PerCP-Cy5.5 (eBioscience, 45-5981-82), hamster anti-CD48 APC (eBioscience, 17-0481-82), and rat anti-CD150 PE-Cy7 (BioLegend, 115914). All flow cytometry analysis was performed on a BD LSR Fortessa flow cytometer with FlowJo v10.7.1 for Mac.

C3H TLR2^−/− and C3H WT mice were infected with B. burgdorferi and sacrificed at days 14, 21, 28, 42, and 49 postinfection. Hearts were perfused with 1× PBS, removed, and cut into fine pieces. Ankles were harvested from each mouse by removing the toes and carefully cutting through the knee joint, particularly to avoid bone marrow contamination. Excess muscle tissue was trimmed to reduce blood contamination. Ankles and hearts from each mouse were placed in appropriately labeled 15-mL conical tubes containing 5 mL 1× PBS + 4% FBS, 75 μL diluted DNaseI (0.03 mg), and 50 μL stock collagenase/dispase. These were placed on a rocker at room temperature for 1 h before being placed into sterile Petri dishes with 5 mL of additional RPMI supplemented with 10% FBS. Ankle tissue was carefully flayed apart using sterile rat tooth forceps. Cells from joints and hearts were strained through a 70-μm filter (BD Falcon) into a 50-mL conical tube. Cells were spun at 300 g, 4°C, 8 min. Supernatant was removed, and cells were washed with 5-mL 1× PBS + 4% FBS three times. Live cells were counted using 3% acetic acid with methylene blue (Stemcell Technologies).

Hindlimb bones were dissected, and the marrow was flushed through a 21‐gauge needle into HBSS⁺, 2% FBS (Invitrogen), and 10 mM HEPES. The cells were passed through a 25‐gauge needle twice and filtered (40‐μm filter, BD Falcon^™, BD Biosciences, 2350 Qume Drive San Jose, CA 95131 ) to ensure a single‐cell suspension. Nucleated cells were counted manually after the lysis of RBCs with 3% acetic acid in methylene blue (STEMCELL Technologies, 570 West Seventh Avenue, Suite 400, Vancouver, BC, V5Z 1B3, Canada). For the colony‐forming assay, 1 × 10⁴ BM mononucleated cells (BM MNCs) or 3 × 10⁴ GFP+ cells were cultured in 35‐mm dishes containing MethylCult 3630 (STEMCELL Technologies) following the manufacturer's protocols. The colonies were counted on day 8.

To assess the ratio between nucleated and non-nucleated cells in the peripheral blood, non-nucleated cells were lysed using an acetic acid (3%) solution with methylene blue (Stem Cell Technologies, France), and the nucleated cells were manually counted.