Ack lysis buffer

CD45.1STEM mice were used as donor mice. Long bones, pelvis and spines were harvested and muscle tissue was removed. Bones were crushed in PBS complemented with 2% FBS and bone marrow cells in suspension were filtered on a 70μm cell strainer. Bone marrow cells were stained by a biotinylated anti-mouse lineage cocktail of antibodies (B220, CD3, CD4, CD8, Mac1, Gr1, Ter119), Sca-1 BV421 and c-Kit BV711 for 45 minutes at 4 degrees. Red blood cells were lysed using the ACK lysis buffer (VWR, 118-156-721) for 5 minutes on ice, cells were washed and then stained with the Streptavidin BUV395 for 15 min at 4 degrees. Cells were washed and suspended in PBS 2% FBS for FACS sorting on the FACS Aria II sorter. 7AAD live/dead dye (BD Pharmingen, 559925) was added just prior sorting to the cells. 3 × 10⁴ LKS were injected into lethally irradiated control and CD73^−/− mice with 0.2 × 10⁶ supporting unlabeled wild-type BM cells.
The same protocol was applied to sort LKS cells from β-actinDsRED (Tg(CAGDsRed*MST)1Nagy/J, The Jackson laboratory #005441) mice for the co-culture assay.

L929 and HEK293T cells were obtained from the American Type Culture Collection (ATCC) and maintained in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, ThermoFisher Scientific; Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; VWR; Radnor, PA, USA). BMDMs were generated by crushing the tibia and femurs of unpooled mice. After lysis of red blood cells with ACK lysis buffer (VWR; Radnor, PA, USA) cells were filtered through a 40 μM cell strainer and grown on petri plates in DMEM containing 10% FBS and 30% L929 conditioned media for 7 days with fresh L929 containing media media being added on day 4 post plating. IL-4 (PeproTech; Rocky Hill, NJ, USA) was used at a concentration of 20ng/mL for in vitro experiments unless otherwise stated. Human U937 monocytic cells were a gift from Dr. Kristin Patrick and maintained in Roswell Park Memorial Institute 1640 Medium (RPMI 1640; Gibco, ThermoFisher Scientific, Waltham, MA, USA) supplemented with 10% FBS. To differentiate U937 monocytes into macrophages, cells were treated with 100ng/mL phorbol 12-myristate 13-acetate (PMA; AmBeed, Arlington Hts, IL, USA) for 48 hours. After that time period the media was changed, and cells were treated similarly as BMBMs. All cells were cultured at 37 °C with 95% humidity and 5% CO₂.

Visceral fat deposits and a 5cm section of the ileum were harvested from mice and washed with PBS before being placed in a 0.2% (2mg/mL) collagenase 2 solution, (Worthington Biochemical; Lakewood, NJ, USA) diluted in RIPA buffer (Gibco, ThermoFisher Scientific; Waltham, MA, USA), under contestant stirring for 60 minutes at 37°C. The isolated cells were filtered through a 70 μM cell strainer and red blood cells were lysed with ACK lysis buffer (VWR; Radnor, PA, USA). Cells were then stained as described above for the bone marrow.