cell culture murine cancer cell lines  (ATCC)

Journal: Cell metabolism

Article Title: Alteration of Tumor Metabolism by CD4+ T Cells Leads to TNF-α-Dependent Intensification of Oxidative Stress and Tumor Cell Death

doi: 10.1016/j.cmet.2018.05.012

Figure Lengend Snippet: (A) Detection of ROS in cultured CT26 cells. CT26 cells were subjected to the indicated culturing conditions for 18 hr. Mafosfamide (maf) or melphalan (mel) was used as chemotherapy. Cells were stained with CM-H2DCFDA and DAPI to evaluate ROS levels and cell viability, respectively. Gating on live cells (DAPI−), histograms show overlay of CM-H2DCFDA curves under different culturing conditions. The MFIs of CM-H2DCFDA are summarized in bar graphs and shown as mean ± SEM.

Article Snippet: EXPERIMENTAL MODEL AND SUBJECT DETAILS Cell Culture Conditions Murine colorectal cancer cell line CT26 (ATCC), mammary carcinoma 4T1 (ATCC) and B-cell lymphoma cell line A20 (ATCC) were cultured in RPMI 1640 (HyClone Laboratories) supplemented with 10% fetal bovine serum albumin (FBS), 1% penicillin/streptomycin (HyClone Laboratories), 1% non-essential amino acids and 1% glutamine (Corning) at 37°C in a 5% CO2 incubator.

Techniques: Cell Culture, Staining

Journal: Cell metabolism

Article Title: Alteration of Tumor Metabolism by CD4+ T Cells Leads to TNF-α-Dependent Intensification of Oxidative Stress and Tumor Cell Death

doi: 10.1016/j.cmet.2018.05.012

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: EXPERIMENTAL MODEL AND SUBJECT DETAILS Cell Culture Conditions Murine colorectal cancer cell line CT26 (ATCC), mammary carcinoma 4T1 (ATCC) and B-cell lymphoma cell line A20 (ATCC) were cultured in RPMI 1640 (HyClone Laboratories) supplemented with 10% fetal bovine serum albumin (FBS), 1% penicillin/streptomycin (HyClone Laboratories), 1% non-essential amino acids and 1% glutamine (Corning) at 37°C in a 5% CO2 incubator.

Techniques: Recombinant, Activation Assay, Lysis, Binding Assay, Transfection, Luciferase, GSH Assay, SYBR Green Assay, Gel Extraction, Plasmid Preparation, Cell Isolation, Mouse Assay, CRISPR, Software

Journal: Cell metabolism

Article Title: Alteration of Tumor Metabolism by CD4+ T Cells Leads to TNF-α-Dependent Intensification of Oxidative Stress and Tumor Cell Death

doi: 10.1016/j.cmet.2018.05.012

Figure Lengend Snippet: (A) Detection of ROS in cultured CT26 cells. CT26 cells were subjected to the indicated culturing conditions for 18 hr. Mafosfamide (maf) or melphalan (mel) was used as chemotherapy. Cells were stained with CM-H2DCFDA and DAPI to evaluate ROS levels and cell viability, respectively. Gating on live cells (DAPI−), histograms show overlay of CM-H2DCFDA curves under different culturing conditions. The MFIs of CM-H2DCFDA are summarized in bar graphs and shown as mean ± SEM. (B) TNF-α synergizes with chemotherapy to induce tumor cell death. Percentage of dead cells under each culturing condition was evaluated by DAPI staining. Results are summarized in bar graph and shown as mean ± SEM. (C) Measurement of GSH and GSH/GSSG ratio in tumor cells. CT26 cells were treated under the indicated conditions for 7 hr. Intracellular GSH and GSSG contents were measured and the GSH/GSSG ratio was calculated. Results are summarized in bar graphs and shown as mean ± SEM. (D) Supplementation of exogenous GSH diminishes cell death induced by the combination of melphalan and TNF-α. CT26 cells were treated by the combination of mel and TNF-α, in the absence or presence of cell-permeable GSHree or mitochondria-selective antioxidant mitoTEMPO. Cell viability was evaluated by DAPI. (E) Loss of TNFR1 leads to reduced tumor cells death after mel + TNF-α treatment. TNFR1 knockout CT26HA cells (CT26HA.ΔTNFR1) and the parental CT26HA cells were subjected to the indicated culturing conditions. After overnight incubation, cell viability was evaluated by DAPI stain. (F–H) (F) Preparation of CM from polyfunctional CD4+ T cells. The schema depicts the experimental procedures. Polyfunctional CD4+ T cells and control CD4+ T cells were prepared following our established protocol. The polyfunctional status of T cells was confirmed by ICS. The expression profiles of TNF-α and IFNγ are shown by dot plots. Conditioned medium from polyfunctional CD4+ T cells (pCD4 CM) or control CD4+ T cells (cCD4CM) were used to culture CT26 cells in the absence or presence or mel. After overnight culture, cells were harvested and examined for intracellular ROS levels by CM-H2DCFDA staining (G) and cell viability by DAPI (H). (I) Addition of GSHree or TNF-α-neutralizing mAb diminishes cell death induced by the combination of mel and pCD4CM. Data shown are representative of three independent experiments with similar results. *p < 0.05, **p < 0.01, ***p < 0.001; n.s., not significant. Error bars indicate SEM of triplicate data. See also Figures S4 and S5.

Article Snippet: Cell Culture Conditions Murine colorectal cancer cell line CT26 (ATCC), mammary carcinoma 4T1 (ATCC) and B-cell lymphoma cell line A20 (ATCC) were cultured in RPMI 1640 (HyClone Laboratories) supplemented with 10% fetal bovine serum albumin (FBS), 1% penicillin/streptomycin (HyClone Laboratories), 1% non-essential amino acids and 1% glutamine (Corning) at 37°C in a 5% CO2 incubator.

Techniques: Cell Culture, Staining, Knock-Out, Incubation, Control, Expressing

Journal: Cell metabolism

Article Title: Alteration of Tumor Metabolism by CD4+ T Cells Leads to TNF-α-Dependent Intensification of Oxidative Stress and Tumor Cell Death

doi: 10.1016/j.cmet.2018.05.012

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Cell Culture Conditions Murine colorectal cancer cell line CT26 (ATCC), mammary carcinoma 4T1 (ATCC) and B-cell lymphoma cell line A20 (ATCC) were cultured in RPMI 1640 (HyClone Laboratories) supplemented with 10% fetal bovine serum albumin (FBS), 1% penicillin/streptomycin (HyClone Laboratories), 1% non-essential amino acids and 1% glutamine (Corning) at 37°C in a 5% CO2 incubator.

Techniques: Virus, Recombinant, Liposomes, Activation Assay, Lysis, Binding Assay, Transfection, Luciferase, GSH Assay, SYBR Green Assay, Gel Extraction, Plasmid Preparation, Cell Isolation, Mouse Assay, CRISPR, Software