F4 80 apc efluor 780

To obtain bone-marrow derived macrophages, femurs from control mice, or mice carrying floxed alleles of Slc2a1 or Glut1^myc knock-in mice were removed and flushed with 5mL of sterile PBS containing 5% FBS^{28 ,29} . The cell suspension was centrifuged, treated with red blood cell lysis buffer, washed, and then plated onto sterile petri dishes in DMEM containing 10% L929 media, 10% FBS and 1% penicillin/ streptomycin/glutamine (PSQ). Media was replenished every 2 to 3 days and differentiated cells were used at day 6 post-harvest. To delete Slc2a1, macrophage cultures were treated with TAT-Cre (EMD Millipore) according to manufacturer’s instructions. For staining, BMDMs were stained with F4/80 APC-eFluor 780 (eBioscience, Cat#: 47–4801-80) and subsequently stained with Myc PerCP antibody (Novus Biologicals, 9E10 Cat#: NB600–302PCP) or fixed and permeabilized using FoxP3/Transcription Factor Staining Buffer Set (eBioscience), and intracellular staining was performed using CD206 PE (BioLegend, Cat#: 141706). Resident peritoneal macrophages were obtained by flushing the peritoneal cavity of mice with 10mL of cold PBS containing 5% FBS. Collected cells were spun down, resuspended in X-VIVO 10 (Lonza), and plated at a concentration of 5×10⁵ cells per well. Floating cells were removed the next day, and remaining peritoneal macrophages were used 2 days after isolation.