Magnisort mouse cd3 positive selection kit

For ex vivo cytotoxicity analysis of ICB R, ICB NR, and C Gl261 TILs, a lactate dehydrogenase (LDH) release assay was applied (Promega; G1780). A total of 5 × 10³ Gl261 cells were seeded onto 96-well flat bottom plates and incubated at 37 °C, 5% CO₂ over night to allow for tumor cell adherence. For isolation and purification of TILs from ICB R, ICB NR, and C-treated Gl261 tumors on day 27 post inoculation, tumor-bearing hemispheres were processed to single-cell suspensions, and TILs were purified with myelin removal beads II (Miltenyi Biotec; 130-096) and MagniSort™ Mouse CD3 Positive Selection Kit (eBioscience; 8802-6840) according to the manufacturer’s instruction. Purified TILs were co-cultured with Gl261 cells at a ratio of 10:1 (5 × 10⁴ CD3⁺T − 5 × 10³ Gl261 cells) at 37 °C, 5% CO₂ for 4 h. A total of 4–5 ICB R, ICB NR, and C TIL samples per group were pooled for cytotoxicity analysis. LDH release of TIL-mediated Gl261 killing was assessed with the CytoTox 96® Non-Radioactive Cytotoxicity Kit (Promega) according to the manufacturer’s instruction and OD was measured on a iMarkTM Microplate reader (BioRad; Hercules, Calif.) at 490 nm. Values were corrected for spontaneous effector and target cell LDH release and cell culture medium background. Data are represented as tumor cell lysis relative to positive Gl261 lysis control.

At day 0 of FV infection, NKT cells were stimulated by i. p. application of 2 µg chemically synthesized αGalCer (KRN7000, Cayman Chemical Company) diluted in PBS. For isolation of NKT cells, CD3⁺ cells were isolated with MagniSort^® Mouse CD3 Positive Selection Kit (eBioscience) and cells were sorted for NK1.1⁺ cells. For transfer experiment, 1 × 10⁵ NKT cells per mouse were diluted in PBS and injected i.v. at the day of FV infection.

The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were determined using a Seahorse XF96 Extracellular Flux Analyzer (Agilent Technologies Inc.) following established protocols18 (link). Briefly, P2RY12^-/- mice and age- and sex-matched WT control mice (8-10 weeks) were used to establish the ConA-induced immune hepatitis model by injecting ConA into tail vein at the dose of 12.5 mg/ kg of body weight for 12 hr. Hepatic lymphocytes isolated and further sorted T cells with Magnisort^TM mouse CD3 positive selection kit (Invitrogen, Carlsbad, CA, USA). OCR was evaluated under basal conditions and in response to 1 μM oligomycin, 0.6 μM FCCP, 100 nM rotenone, plus 1 μM antimycin A. ECAR was measured under basal conditions and in response to 100 mM glucose, 1 μM oligomycin, and 5 mM 2-d-glucose (2-DG). Activated T cells were cultured in XF-medium (XF Base Medium Minimal Dulbecco's modified Eagle's medium (DMEM); Agilent Technologies Inc.) containing 10 mM glucose, 2 mM L-glutamine, and 1 mM sodium pyruvate. Assays were analyzed with the Wave Desktop software (Agilent Technologies Inc.).