Nh4cl

Mice were deeply anesthetized with 5% isoflurane (Aerrane, Baxter) and transcardially perfused with 50 mL of cold phosphate-buffered saline, pH 7.4 (PBS, Sigma-Aldrich). Spinal cords were harvested at the EAE plateau phase [day post-immunization (dpi) 20 ± 3] and homogenized in PBS. Leukocytes were recovered at the 30:70% Percoll (Fisher Scientific) interface after gradient centrifugation as described in the literature [23 ] and were then counted with the Malassez chamber. Spleens were aseptically removed from naïve and MOG-immunized C57BL/6 mice at the peak of clinical score (≥ 3, dpi 15–18), as described previously [24 (link)], mechanically processed to obtain a splenocyte suspension by passing the cells through a 40-μm filter (Falcon). Erythrocytes were lysed in lysis buffer [0.15 M NH4Cl, 9 mM HKCO3, 0.5 M EDTA, pH 7.4 (Stemcell Technologies)], and the sterile splenocytes were resuspended in supplemented sterile PBS with 10% filtered and inactivated fetal bovine serum (FBS, Stemcell Technologies), 1% Penicillin/Streptomycin (Gibco), and 2.5% (v/v) HEPES (Fisher).

BM from the tibiae and femurs of each mouse were flushed. Red blood cells (RBCs) were lysed for 5 min at 4 °C in 0.15 M NH₄Cl (STEMCELL Technologies, Vancouver, Canada), washed with PBS (Gibco), and counted using a hemocytometer. For HSC, MSC, or osteoblast detection, Lin⁺ cells were removed by magnetic depletion using biotinylated lineage-specific antibodies (CD5, CD45R, CD11b, Gr-1, and Ter-119), followed by depletion with MACs beads conjugated to monoclonal anti-biotin (Miltenyi Biotec, Gladbach, Germany). For staining of HSCs, Lin⁻ cells were stained with phycoerythrin (PE)-Cy7-conjugated antibodies to Sca1 (558162), APC-conjugated antibodies to c-Kit (553356), FITC-conjugated antibodies to CD48 (557484), and PE-conjugated antibodies to CD150 (561540), all from BD Biosciences. Cells were further stained with streptavidin-pacific blue (PB) (Invitrogen, S11222). Data were collected using AriaIII systems (BD Biosciences) and analyzed using FlowJo software (Tree Star, Ashland, OR).

Cells were isolated from the tibia and femur and crushed once, using mortar and pestle, then cut into small pieces with scissors. The tissue was subjected to three rounds of enzymatic digestion with 0.2% collagenase at 37 °C under agitation for 30 min each. Cells were filtered through a 70 μm strainer and centrifuged at 1 500 r·min⁻¹ for 5 min at 4 °C. Red blood cells were lysed using NH₄Cl (StemCell Technologies, Vancouver, Canada) for 10 min on ice, washed with staining media (HBSS (Thermo Fisher Scientific, Waltham, MA, USA) containing 2% fetal bovine serum (Life Technologies: 10437-028), 1% HEPES (10 mmol·L⁻¹) (Thermo Fisher Scientific) and 1% penicillin-streptomycin (Thermo Fisher Scientific) and resuspended in HBSS for subsequent analyses.

Mammary glands were dissected from 10- to 12-week-old female mice at pregnancy day 14.5, cut into pieces, and placed in DMEM/F12 (Gibco) with 5% FBS (Gibco), 300 U/mL collagenase (Gibco) and 100 U/mL hyaluronidase (Sigma) for 2 h at 37°C. After digestion, red blood cells were removed with NH₄Cl (STEMCELL Technologies, Vancouver, BC, Canada), and then a single-cell suspension was obtained by sequential dissociation in 37°C preheated 0.25% trypsin (Gibco) for 2 min and in 5 mg/mL Dispase (STEMCELL Technologies) containing 0.1 mg/mL DNase I (Sigma) for 5 min with pipetting, followed by filtration through a 40 μm filter (BD, San Jose, CA, United States). The single-cell suspension was stained with specific antibodies in DPBS containing 2% FBS for 15 min on ice. After being washed three times with DPBS containing 2% FBS, cells were analyzed by FACS Verse or sorted by FACS Aria. The following antibodies were used: CD45-APC (17-0451-82, eBioscience, San Diego, CA, United States), CD31-APC (17-0311-82, eBioscience), TER119-APC (17-5921-82, eBioscience), CD24-PE-Cy7 (25-0242-82, eBioscience), CD29-FITC (11-0291-82, eBioscience), CD61-PE (561910, BD), and Fixable Viability Dye eFluor 450 (65-0863-14, eBioscience). Data analysis was performed with FlowJo 7.6.1.

Tumor cells were isolated from transgenic mammary tumors as previously described. Briefly, dissected tumors were minced into fragments under sterile conditions and subjected to enzymatic digestion with Collagenase-Type3 (Worthington, Cat#: LS004183) at 2 mg/ml, Dispase (Gibco, Cat#: 17105-041) at 2 mg/ml, Gentamycin (Gibco, Cat#:15710-064) at 50 µg/ml, Amphotericin B (Sigma, Cat#:A2942) at 250 µg/ml, and 5% FBS in DMEM/F12 media for 3 h with intermittent shaking. Following digestion, tumor organoids were pelleted and then treated with NH4Cl (Stem Cell Technologies, Cat # 07800) to lyse RBCs, following which, the pellet was washed three times with PBS. Organoids were then passed through a 70 µm cell strainer, counted and used for transplantation experiments or cultured at a density of 3 × 10⁶ cells/55cm². Proliferation of cultured cells was measured by assessing BrdU incorporation (Cell proliferation ELISA Kit 11647229001; Roche) after addition of Dox (2 µg/ml) or PTHrP (Bachem, Cat# 4017147.0500) to the culture media.