Clone 2.4g2

Single-cell suspensions were prepared from WAT. Cells were first incubated with anti-mouse CD16/32 antibody to prevent non-specific binding (BD Biosciences, clone: 2.4G2). Then cells were stained with fluorescence-conjugated antibodies (Supplemental Experimental Methods and Materials) in FACS buffer, washed, and resuspended in FACS buffer with propidium iodide (Sigma-Aldrich P4684). Flow cytometry experiments were performed on BD LSR II (BD Biosciences) and analyzed using FlowJo software.

Blood samples (~120 μL) were withdrawn from the facial vein of C57BL/6 (WT) recipient mice 8 weeks after transplantation with GFP⁺ bone marrow cells (GFP^+/-→WT) (n = 15) as well as of GFP^+/- (n = 9) and WT (n = 6) animals. Samples were quickly collected in EDTA-coated tubes (Starstedt, Montreal, Quebec, Canada) to prevent coagulation. A volume of 35 μL of DPBS without Ca²⁺ and Mg²⁺ (Sigma-Aldrich, St Louis, MO) was added to 65 μL of blood and incubated for 20 min on ice with purified rat anti-mouse CD16/CD32 antibody diluted 1:100 (clone 2.4G2; BD Biosciences) to block non-specific binding of IgGs to Fc receptors. Samples were washed and resuspended in 100 μL of DPBS after being centrifuged at 300 x g for 10 min. Cell suspensions were then labeled with the following rat anti-mouse antibodies for 40 min at 4°C: PE-Cy5-CD45 (clone 30-F11; BD Biosciences), APC-CD115 (clone AF598; eBioscience, San Diego, CA), PE-Cy7-CD11b (clone M1/70; eBioscience), V450-Ly6C (clone AL21; BD Biosciences) and PE-Ly6G (clone 1A8; BD Pharmingen, San Jose, CA). Red blood cells were lysed with BD Pharm Lyse™ (BD Biosciences) during 30 min at room temperature, and the recovered leukocytes were washed and resuspended in DPBS for analysis. Flow cytometry analysis and data acquisition were performed using a BD SORP LSR II and the BD FACSDiva software, respectively.

Whole blood samples were obtained from the anaesthetized mice using a Goldenrod animal lancet (5 mm point size, Braintree Scientific Inc.). The blood sample was collected from the back of the jaw and were transferred into EDTA-coated collection tubes. For fluorescence-activated cell sorting (FACS) analysis, whole blood samples were treated with RBC lysis buffer (Sigma-Aldrich) for 10 min at rt. Then, an equal amount of PBS was added before centrifuging the samples at 300×g at rt. The resulting cell pellet was washed with PBS-1%BSA and resuspended in PBS-1%BSA containing Fc-Block (1/200; Clone 2.4G2; BD Pharmingen). The antibody was incubated at 4 °C for 30 min. Antibodies used for cell staining are as follows: anti-NK1.1-BV421 (1:200, 108741, BioLegend), anti-B220-PE/Cy7 (1:200, 103222, BioLegend), anti-CD3-FITC (1:200, 100204, Biolegend), anti-CD11b-APC (1:200, 101211, Biolegend), anti-CD11c-PE (1:200, 117307, BioLegend).