Collagenase 4

The infiltration of lymphocytes in tumor tissue was analyzed by flow cytometry, as described elsewhere (20 (link)). To generate single cell from the tumor tissue (100-200 mg), Collagenase IV (Yeasen Biotech, Shanghai, China) was employed to digest the tissue in HBSS at 37°C for 30 minutes. The single cells were obtained by filtering the mixture through a 70 μm cell strainer (BD, Franklin Lakes, USA). Then, surface staining with anti-mouse antibodies for CD45 (GK1.5), CD4 (30-F11) and CD8 (53-6.7) was carried out on the single cells in the dark at 4°C for 30 min. After washing twice with PBS, the stained cells were fixed in a Foxp3/Transcription Factor Staining Buffer Set (eBioscience, San Diego, USA) in the dark at 4°C for 30 min. Subsequently, the fixed cells were permeabilized and stained with antibodies for IFN γ (XMG1.2) in the dark at 4°C for 30 min. Finally, the cells were washed twice, suspended in fluorescence-activated cell sorting (FACS) buffer, and analyzed using a FACS Calibur flow cytometer (BD, Franklin Lakes, USA). FlowJo software (TreeStar, Ashland, USA) was utilized to interpret the results.

Oligomycin, cyanide p-trifluoromethoxyphenyl-hydrazone (FCCP) and rotenone were purchased from Seahorse Biosciences, USA; 2-Deoxy-D-glucose, glucose, palmitic acid (Palm.), rapamycin were purchased from Sigma-Aldrich, USA; Recombinant IL-22 proteins were obtained from Novoprotein, China; dorsomorphin, LY294002, cisplatin (Cisp.), 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1), TRNzol reagent, MitoTracker Green, SYBR Green qPCR mix, MMLV reverse transcriptase were provided by Beyotime Biotechnology, China; ethanol (Eth.), carbon tetrachloride (CCl₄) were obtained from Sinopharm, China; MitoTracker Red, Hoechst33342, PKH26, MitoSOX were purchased from Invitrogen, USA. Collagenase IV was obtained from Yeasen, China. Percoll was purchased from GE, USA. Antibodies targeting AKT, p-AKT (S473), mTOR, p-mTOR (S2448), p-STAT3 (Y705), p-PI3K (Tyr199), p-p70S6K, HK-2, c-Myc, Bad, Bcl-2 were obtained from Cell Signaling Technology, USA; Antibodies for Glu1, AMPK, p-AMPKα, IL-22R1, β-Actin and GAPDH were provided by Abcam, USA.

The intestines were incubated in HBSS containing 5  mM EDTA at 37 °C with shaking for 20 min [1 (link),26 (link)], and then cut into small pieces, digested with RPMI 1640 containing 10% FBS, collagenase IV (100  mg/mL, Yeasen, Shanghai, China), DNase I (50 mg/mL, Yeasen) at 37 °C for 1 h in a thermostatic oscillator. Fresh heart tissues were washed with cold PBS and cut into small pieces, then digested with RPMI 1640 containing 10% FBS, collagenase II (50 U/μL, Gibco, New York, NY, USA), and hyaluronidase (30 mg/mL, Sigma, Livonia, MI, USA) at 37 °C for 1 h.
The digested tissues were filtered through a 70 μm cell strainer. The cells were resuspended in a discontinuous 40/80% Percoll gradient at 500 g for 30 min. The immune cells were collected at the interface of the Percoll gradient.