Human peripheral blood T cell subsets (106/ml) were activated with anti-CD3 (clone UCHT1, 2.5 μg/ml) and anti-CD28 (clone CD28.2, 1.25 μg/ml) antibodies (BD Bioscience) for 3–5 days, as indicated in the figure legends. To obtain tumor medium, primary ovarian cancer cells were cultured for 3 days, and subsequently were frozen and thawed 5 times along with the culture medium. The cancer cell culture supernatant was obtained through centrifugation (20,000g, 1 h, 4°C), and used to culture T cells with or without glucose (2 mg/ml, Sigma Aldrich) supplementation for 16–24 h. To obtain normal medium containing different concentrations of glucose, normal medium without glucose (Invitrogen) was supplemented with 1 or 5 mg/ml glucose. Chemical inhibitors of metabolic pathways, Notch signaling or EZH2 were added as indicated in figure legends. Total numbers of viable cells at the endpoint of experiments were 1–2 × 106 cells.
Normal medium without glucose
Manufactured by Thermo Fisher Scientific
Normal medium without glucose is a cell culture medium that does not contain the carbohydrate glucose. It provides the necessary nutrients and components required for the growth and maintenance of cells in vitro, excluding glucose as an energy source.
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2 protocols using normal medium without glucose
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Tumor-Induced T Cell Metabolism Modulation
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Tumor-Induced T Cell Metabolism Modulation
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Human peripheral blood T cell subsets (106/ml) were activated with anti-CD3 (clone UCHT1, 2.5 μg/ml) and anti-CD28 (clone CD28.2, 1.25 μg/ml) antibodies (BD Bioscience) for 3–5 days, as indicated in the figure legends. To obtain tumor medium, primary ovarian cancer cells were cultured for 3 days, and subsequently were frozen and thawed 5 times along with the culture medium. The cancer cell culture supernatant was obtained through centrifugation (20,000g, 1 h, 4°C), and used to culture T cells with or without glucose (2 mg/ml, Sigma Aldrich) supplementation for 16–24 h. To obtain normal medium containing different concentrations of glucose, normal medium without glucose (Invitrogen) was supplemented with 1 or 5 mg/ml glucose. Chemical inhibitors of metabolic pathways, Notch signaling or EZH2 were added as indicated in figure legends. Total numbers of viable cells at the endpoint of experiments were 1–2 × 106 cells.
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