Quadromacs magnet

eWAT SVF was obtained from n=3 C57BL/6J mice as described above and pooled for each independent experiment. RBC-depleted SVF cells were washed in 1 mL MACS Buffer (PBS containing 1% bovine serum albumin and 1 mM EDTA) and pelleted by centrifugation at 500 x g for 5 min at 4 °C. The cells were resuspended in 90 μL of MACS Buffer followed by addition of 10 μL magnetic nanobeads conjugated to anti-F4/80 antibody (Miltenyi Biotec). After a 15-min incubation at 4 °C covered in foil, the SVF cells were passed through a 30 μm nylon mesh and then an LS Column on a QuadroMACS magnet (Miltenyi Biotec). Cells were washed 3 times with 3 mL of MACS Buffer and then eluted in 5 mL MACS Buffer. Purity was confirmed by flow cytometry and was >95%. The cells were pelleted by centrifugation at 500 x g for 5 min at 4 °C, and the cells were resuspended in 2 mL Wash Buffer containing 10 μM CytoTracker Orange (also known as CMTMR; ThermoFisher; stock solution 10 mM in DMSO) and incubated at 37 °C for 30 min. The CMTMR-labelled cells were washed 2 times in 25 mL Wash Media and one time in 25 mL PBS. The washed cell pellet was resuspended in 300 μL PBS for cell counting. Cells (approximately 5 x 10⁴ per mouse) were administered by intraperitoneal injection into male or female mtD2 mice or male MitoFat mice and then imaged by intravital 2-photon microscopy 1-3 days after the adoptive transfer.

Mitochondria were isolated using the Mitochondria Isolation Kit, Mouse Tissue (Miltenyi Biotec). mtDendra2 mice were euthanized, the right atrium was punctured, and the mouse was perfused with 10 mL PBS (sterile, ice-cold) via the left ventricle. Perfused liver was harvested, finely minced, and homogenized in a 2 mL dounce homogenizer in 1 mL Lysis Buffer with 7-8 strokes. Tissue homogenate was transferred to a 15 mL conical tube containing 9 mL 1X Separation Buffer (SB) and mixed thoroughly. Anti-TOM40 antibody conjugated to magnetic nanobeads (50 μL) was added and incubated in the dark at 4 °C with gentle rocking for 1 hour. The mixture was passed through a 30 μm nylon mesh and then an LS Column associated with a QuadroMACS magnet (Miltenyi Biotec). The column was washed 3 times with 3 mL of 1X SB. Purified mitochondria were eluted with 1.5 mL of 1X SB and pelleted by centrifugation at 15,000 x g for 2 min at 4 °C. The pellet was resuspended in 1 mL Storage Buffer (Miltenyi Biotec), pelleted again as above, resuspended in 1 mL Storage Buffer, and held on ice. Mitochondria protein concentration was determined using 10 μL of purified mitochondria and 300 μL Coomassie Plus Reagent (Pierce) alongside a standard curve of Bovine Serum Albumin (BSA, Piece). Absorbance was measured at 450 nm using a BioRad iMark Microplate Reader.

Tumour tissues were carefully isolated 24 h after the final treatment (Day 20) and dissociated as described above. Macrophages or DCs were isolated by using F4/80 or CD11c Microbeads, respectively, on a QuadroMACS magnet (Miltenyi) according to the manufacturer’s instructions. Total RNA was extracted from the obtained cell populations by using a PureLink RNA Mini Kit (Applied Biosystems), and cDNA was prepared with High-Capacity cDNA RT Kit (ThermoFisher) according to the manufacturer’s instructions. RT-qPCR was done with the 7500 FAST Real-time PCR System (Applied Biosystems), with TaqMan^™ Fast Advanced PCR master Mix (ThermoFisher) and TaqMan FAM dye-labelled probes including GAPDH (Hs02786624_g1, Mm99999915_g1), IFNA1 (Mm03030145_gH), and IFNB1 (Mm00439552_s1) (ThermoFisher). GAPDH was used to normalise signal expression. Fold-change comparisons were done between control and treated samples by using the ΔΔCT method as described elsewhere⁴⁹ .