Lineage cell detection cocktail biotin

CL429 (Cat. Code: vac‐c429) was purchased from InvivoGen. Lipopolysaccharide (LPS) from Escherichia coli was purchased from Sigma (St.Louis, MO, USA), and Pam3CSK4 was obtained from EMC Microcollection (Tübingen, Germany). The TransDetect Annexin V‐FITC/PI Cell Apoptosis Detection Kit was purchased from TransGen Biotech (Beijing, China). RPMI 1640 and foetal bovine serum (FBS) were supplied by Gibco. Streptavidin FITC, anti–Mouse CD117 APC eFluor 780, and anti‐Mouse Ly‐6A/E (Sca‐1) PerCP‐Cyanine5.5 were purchased from BD Pharmingen (San Diego, CA). Lineage Cell Detection Cocktail‐Biotin was purchased from Miltenyi Biotec (Germany). Lipofectamine 3000 was purchased from Thermo Fisher. Antibodies were purchased from Affinity Biosciences (Jiangsu, China) and Cell Signaling Technology (Massachusetts, USA). Commercial ELISA kits were purchased from DAKEWE (Beijing, China).

Bone marrow cells, which were freshly isolated from tibia and femur of male and female young (3 to 6 month) and old (18 to 24 month) C57BL/6 mice, were enriched by magnetic activated cell separation (Miltenyi Biotech, Bergisch Gladbach, Germany), immunolabeled with Sca-1 and lineage antibodies and sorted by FACS^{118 (link)}. In brief, crushed bone samples were incubated with allophycocyanin (APC)-conjugated anti-mouse c-Kit (clone 2B8; 1:100; # 17-1171; Thermo Fisher Scientific) for 30 min at 4 °C and combined after washing with anti-APC microbeads (Miltenyi Biotech). cKit⁺ cells were magnetically enriched using a LS column (Miltenyi Biotech) and incubated with lineage cell detection cocktail-biotin (# 130-092-613, Miltenyi Biotech) for 30 min at 4 °C and with APC anti-mouse c-Kit (clone 2B8; 1:100; # 17-1171; Thermo Fisher Scientific), PE/Cy7 anti-mouse Sca-1 (clone E13-161.7; 1:100; # 122513; BioLegend, San Diego, CA), and APC/Cy7-conjugated streptavidin (1:100; # 405208; BioLegend) overnight at 4 °C. After staining with DAPI, Lin^- cKit⁺ ScaI⁺ (KSL) cells and Lin^- cKit⁺ ScaI^- myeloid progenitor (MP) cells were sorted using a FACSAria II instrument (BD Biosciences, Franklin Lakes, NJ). FlowJo software was used for data analysis.

Hind limb bones were crushed in a mortar and total BM cells were filtrated (30 µm). Magnetically lineage-depleted BM cells (Lineage Cell Detection Cocktail-Biotin, 130-092-613, Miltenyi Biotec) were stained in phosphate-buffered saline (PBS), pH7.2, with combinations of antibodies for flow cytometry. For the transplantation study, Ly.2 and Ly.1 mice were fed with a CD or HFD, respectively, for 4 weeks and sacrificed for BM transplantation (200,000 cells). Cells were transplanted in competition into the tail vein of lethally irradiated (900 cGy) recipients. PB and BM reconstitutions were analyzed 16 weeks after the transplantation. Further information regarding transplantation were described on the supplementary Methods section.