EL4 and EG7-OVA cells (ATCC) were grown in RPMI 1640 supplemented with 10% FBS and 25mM HEPES (Gibco). EG7-OVA cultures were supplemented with G418 (0.4 mg/ml, InvitroGen). CD8 T cells were isolated from OT1 (Jackson Labs) splenocytes by MACS using CD8a Microbeads (Miltenyi). Cells were activated by seeding in 96-well plates pre-coated with anti-mouse CD3e (1 μg/ml working concentration, Clone: 145–2C11, BD) and anti-mouse CD28 (2 μg/ml working concentration, Clone: 37.51, BD) at 2×106 cells/ml in RPMI 1640 supplemented with 10% FBS, 100U/ml penicillin-streptomycin, 1X non-essential amino acids (Gibco), 1mM sodium pyruvate, 0.05mM 2-mercaptoethanol, and 30U/ml hIL-2 (Roche). After 2 days, cells were washed and transferred to uncoated plates. On day 5, 1 × 106 activated OT1 T cells were coincubated with 1×106 EL4 or EG7-OVA cells for 2 hours at 37 °C and stained for GzmB using anti-mouse GzmB (Clone: NGZB, eBioScience) and Intracellular Fixation & Permeabilization Buffer Set (eBioScience, 88–8824-00). To measure GzmB activity inside target cells, we coincubated activated OT1 CD8 T cells with EL4 and EG7-OVA target cells at various T cell to target cell ratios and stained using GranToxiLux Kit (OncoImmunin, GTL702–8). To measure secretory GzmB, we collected coculture supernatant of OT1 with target cells and performed ELISA with Granzyme B Mouse ELISA Kit (eBioScience, BMS6029).
E g7 ova cell
Manufactured by American Type Culture Collection
Sourced in United States, Japan
The E.G7-OVA cells are a mouse T cell lymphoma cell line that expresses the ovalbumin antigen. They are commonly used in research related to immune responses and cancer immunology.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Intracellular fixation permeabilization buffer set, by Thermo Fisher Scientific (2 mentions)
Cd8a microbeads, by Miltenyi Biotec (2 mentions)
Anti mouse cd28, by BD (2 mentions)
Anti mouse gzmb, by Thermo Fisher Scientific (2 mentions)
Granzyme b mouse elisa kit, by Thermo Fisher Scientific (2 mentions)
Anti mouse cd3e, by BD (2 mentions)
Hepes, by Thermo Fisher Scientific (2 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Hil 2, by Roche (2 mentions)
C57bl 6j mice, by Beijing Huafukang Biotechnology (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 medium, by American Type Culture Collection (2 mentions)
Anti mouse ifn γ, by BioLegend (1 mentions)
Facs aria cell cytometer, by BD (1 mentions)
Anti mouse cd8α, by BioLegend (1 mentions)
Anti mouse cd4, by BioLegend (1 mentions)
α mem, by Thermo Fisher Scientific (1 mentions)
Complete rpmi 1640, by Thermo Fisher Scientific (1 mentions)
19 protocols using e g7 ova cell
1
Quantifying Cytotoxic T Cell Responses Against Tumor Antigens
2
Immunotherapy for Melanoma in Mice
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E.G7-OVA cells (2 × 105 cells/mouse, ATCC, Manassas, VA, USA) were injected subcutaneously into the right flank of C57BL/6 mice (6 weeks old, female, CLEA Inc.). On days 7, 10, 14, and 21, mice were divided into 4 groups, and saline, OVA (100 μg/mouse), OVA-TLR7a (100 μg/mouse for OVA; 20 μg/mouse for Imiquimod), or Gd2O3-OVA-TLR7a (100 μg/mouse for OVA; 20 μg/mouse for Imiquimod; 1 mg/mouse for Gd2O3 nanotubes) were injected into their left flanks. The tumor volume was calculated by 1/2 × longest dimension × (perpendicular dimension)2. Mouse survival rate was calculated on the basis of tumor size < 15 mm. Splenocytes were collected, stained with anti-mouse CD4, anti-mouse CD8α, anti-mouse IFNγ, and anti-mouse TNFα antibodies (BioLegend, San Diego, CA, USA), and analyzed using a FACSAria cell cytometer (BD Biosciences).
3
Quantifying Cytotoxic T Cell Responses Against Tumor Antigens
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EL4 and EG7-OVA cells (ATCC) were grown in RPMI 1640 supplemented with 10% FBS and 25mM HEPES (Gibco). EG7-OVA cultures were supplemented with G418 (0.4 mg/ml, InvitroGen). CD8 T cells were isolated from OT1 (Jackson Labs) splenocytes by MACS using CD8a Microbeads (Miltenyi). Cells were activated by seeding in 96-well plates pre-coated with anti-mouse CD3e (1 μg/ml working concentration, Clone: 145–2C11, BD) and anti-mouse CD28 (2 μg/ml working concentration, Clone: 37.51, BD) at 2×106 cells/ml in RPMI 1640 supplemented with 10% FBS, 100U/ml penicillin-streptomycin, 1X non-essential amino acids (Gibco), 1mM sodium pyruvate, 0.05mM 2-mercaptoethanol, and 30U/ml hIL-2 (Roche). After 2 days, cells were washed and transferred to uncoated plates. On day 5, 1 × 106 activated OT1 T cells were coincubated with 1×106 EL4 or EG7-OVA cells for 2 hours at 37 °C and stained for GzmB using anti-mouse GzmB (Clone: NGZB, eBioScience) and Intracellular Fixation & Permeabilization Buffer Set (eBioScience, 88–8824-00). To measure GzmB activity inside target cells, we coincubated activated OT1 CD8 T cells with EL4 and EG7-OVA target cells at various T cell to target cell ratios and stained using GranToxiLux Kit (OncoImmunin, GTL702–8). To measure secretory GzmB, we collected coculture supernatant of OT1 with target cells and performed ELISA with Granzyme B Mouse ELISA Kit (eBioScience, BMS6029).
4
OP9 Cell Line Generation and Maintenance
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OP9 cells were a kind gift from Dr. Hiroshi Kawamoto (Kyoto University). Control OP9 feeder cells, mouse Delta-like1 expressing-OP9 cells (OP9-DL1), and human Delta-like 1 expressing-OP9 cells (OP9-hDL1) were cultured in alpha MEM (Thermo Fisher Scientific) supplemented with 20% FBS and 1% penicillin/streptomycin. E.G7-OVA cells were purchased from ATCC (Manassas, VA USA). E.G7-OVA cells were cultured in complete RPMI 1640 (Thermo Fisher Scientific) supplemented with 10% FBS, 1% penicillin/streptomycin, and 400 μg ml−1 G418 (Nacalai Tesque, Kyoto, Japan). Lymphoblastoid cell lines (LCLs) were established from peripheral blood mononuclear cells (PBMCs) provided from three healthy donors, as described in previous studies25 (link)26 . PBMCs were cultured in complete RPMI medium with 10% FBS, Epstein–Barr virus (EBV) solution, and 20 nM FK506 for more than one month.
5
Efficacy of SNS-101-m2 in Subcutaneous Tumors
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EG.7-OVA cells (1 × 106 cells per animal; ATCC CRL-2113) were injected subcutaneously into female hVISTA-KI mice (n = 8 mice/group). When tumors reached ~60–100 mm3, animals were randomized and treated thrice weekly for 3 weeks via i.p. injections of either isotype control IgGs, SNS-101-m2, or α-mouse CTLA-4 (clone 9H10, Bio X Cell BE0131). Tumor volumes were measured three times per week. Statistical significance in growth inhibition was determined using Mann-Whitney unpaired t-test, with P < 0.05 considered to be statistically significant.
6
Tumor Rejection Efficacy Evaluation
7
Murine Lymphoma Cell Culture Protocol
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BMDCs were prepared as reported previously15 (link),39 (link). The detail procedure is described in the Supplementary Information. E.G7-OVA cells, a murine lymphoma cell line EL4 expressing OVA, were purchased from the American Type Culture Collection (Manassas, VA) and were cultured in RPMI 1640 medium containing 50 μM 2-mercaptoethanol, 10 mM HEPES, 1 mM sodium pyruvate, 100 U/mL penicillin-streptomycin, 400 μg/mL G418 and 10% fetal bovine serum (FBS).
Female C57BL/6J mice (6–9 weeks old) were purchased from Japan SLC Inc. (Shizuoka, Japan) and maintained under specific pathogen-free conditions. The use of the mice was approved by the Ethics of Pharmaceutical Science Animal Committee of Hokkaido University (approval number: 16-0014). All experiments were performed in accordance with National University Corporation Hokkaido University Regulations on Animal Experimentation.
Female C57BL/6J mice (6–9 weeks old) were purchased from Japan SLC Inc. (Shizuoka, Japan) and maintained under specific pathogen-free conditions. The use of the mice was approved by the Ethics of Pharmaceutical Science Animal Committee of Hokkaido University (approval number: 16-0014). All experiments were performed in accordance with National University Corporation Hokkaido University Regulations on Animal Experimentation.
8
Murine Dendritic Cell and Tumor Cell Co-culture
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DC2.4 murine dendritic cells (obtained from the Third Military Medical University) and EG7-OVA cells (obtained from American Type Culture Collection) were both cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and streptomycin-penicillin (1%, v/v) at 37 °C in a humidified atmosphere incubator containing 5% CO2. Female C57BL/6 mice (6–8 weeks old) were purchased from Chengdu Dossy Experimental Animals Co., Ltd. The mice were kept in a standard animal room, with strict temperature and humidity control, light diurnal cycle, and free access to standard feed and water. All animal experiments were conducted according to the guidelines approved by the Institutional Animal Care and Ethics Committee of Sichuan University (Chengdu, China).
9
Murine Lymphoma Model for OVA Antigen
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E.G7-OVA cells, the murine lymphoma cell line EL4 expressing chicken OVA, were purchased from the American Type Culture Collection (Manassas, VA) and were cultured in RPMI 1640 medium containing 50 μM 2-mercaptoethanol, 10 mM HEPES, 1 mM sodium pyruvate, 100 units/mL penicillin-streptomycin and 10% fetal bovine serum (FBS).
Female C57BL/6J mice (6-8 weeks old) were purchased from CLEA Japan Inc.
(Tokyo, Japan) and maintained under specific pathogen-free conditions. The use of the mice was approved by the Pharmaceutical Science Animal Committee of Hokkaido University.
Female C57BL/6J mice (6-8 weeks old) were purchased from CLEA Japan Inc.
(Tokyo, Japan) and maintained under specific pathogen-free conditions. The use of the mice was approved by the Pharmaceutical Science Animal Committee of Hokkaido University.
10
Culturing Bone Marrow-Derived Dendritic Cells
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Roswell Park Memorial Institute (RPMI) 1640 medium and Dulbecco’s modified Eagle’s medium (DMEM) containing 100 units/ml streptomycin and penicillin (PS) and 10% fetal bovine serum (FBS) were used to culture bone marrow-derived DCs (BMDCs), LL2 cells and EG7-OVA cells (American Type Culture Collection, Manassas, VA, USA). All cells were cultured in a cell incubator containing 5% CO2 at 37 °C. RPMI 1640, DMEM, FBS and PS were all purchased from Thermo Fisher Scientific. Female six- to eight-week-old C57BL/6 J mice were purchased from HFK Bioscience (Beijing, China).
BMDCs were obtained from 4- to 6-week-old C57BL/6 J female mice according to a previously reported protocol [48 (link)]. Briefly, after treating bone marrow cells with red blood cell lysis buffer, fresh RPMI 1640 complete medium containing 20 ng/ml granulocyte-macrophage colony-stimulating factor (GM-CSF, PrimeGene Biotechnology, Shanghai, China) was added to 3 × 106 mouse bone marrow cells. At day 8, the BMDCs were collected for further use.
BMDCs were obtained from 4- to 6-week-old C57BL/6 J female mice according to a previously reported protocol [48 (link)]. Briefly, after treating bone marrow cells with red blood cell lysis buffer, fresh RPMI 1640 complete medium containing 20 ng/ml granulocyte-macrophage colony-stimulating factor (GM-CSF, PrimeGene Biotechnology, Shanghai, China) was added to 3 × 106 mouse bone marrow cells. At day 8, the BMDCs were collected for further use.
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