All cell lines used for in vivo studies were grown and maintained by Charles River Laboratories according to ATCC guidelines and kept in culture for no longer than 2 weeks. Frozen cells were thawed and maintained for one to three passages before implantation. The MC38 cell line was obtained from the NCI Fredrick Cancer DCT Tumor Repository in 2012, the EMT-6 cell line was obtained from ATCC in 2014, the CT26 cell line was obtained from ATCC in 2017, and the B16-F10 cell line was purchased from ATCC in May of 2019. All cell lines were tested for Mycoplasma every 2 months and have not been reauthenticated within the past year. B16-F10, CT26, and EMT-6 cell lines were cultured in RPMI-1640 with L-Glutamine (Gibco, 11875–085) with 10% heat-inactivated FCS (Gibco, 35–015-CV), whereas MC38 was cultured in DMEM (Gibco, 1966–025) supplemented with 10% heat-inactivated FCS (Gibco, 16000–044). Prior to tumor implantation, cells were washed twice with PBS and counted.
Mc38 cell line
Manufactured by American Type Culture Collection
Sourced in United States, France
The MC38 cell line is a murine colon adenocarcinoma cell line. It is a commonly used cancer cell line in research.
Automatically generated - may contain errors
Lab products found in correlation
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
B16f10 cell line, by American Type Culture Collection (1 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 with l glutamine, by Thermo Fisher Scientific (1 mentions)
Ct26 cell line, by American Type Culture Collection (1 mentions)
Emt6 cell line, by American Type Culture Collection (1 mentions)
Endothelial cell growth supplement (ecgs), by ScienCell (1 mentions)
B16 cell line, by American Type Culture Collection (1 mentions)
Penicillin streptomycin, by ScienCell (1 mentions)
T4 dna ligase, by New England Biolabs (1 mentions)
Anti cd8 apc cy7, by BioLegend (1 mentions)
Cd11c apc, by BioLegend (1 mentions)
Dextramer pe, by Immudex (1 mentions)
Pd 1 bv421, by BioLegend (1 mentions)
Phi29 dna polymerase, by New England Biolabs (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
11 protocols using mc38 cell line
1
Cell Line Maintenance and Implantation
2
Optimized Plasmid Constructs for Cancer Immunotherapy
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All constructs were completely synthesized and optimized for codon usage. Synthesis and codon optimization analysis of plasmid DNA vectors were performed at Genscript (China). Luciferase gene, cloned into the linearized pcDNA3-Hygro vector by enzymatic restriction, was used to assess gene expression both in vitro (by transfecting cells) and in vivo (by mice immunization).
conTRT vaccine consisted of a DNA plasmid encoding a consensus sequence between canine and human telomerase. In addition, this catalytically inactive telomerase protein has been fused to a TPA (human tissue plasminogen activator) leader sequence at N-term and fused itself to the Profilin-like sequence of Toxoplasma gondii (PFTG) at the C-terminus (TPA-conTRT-PFTGopt). This construct was finally cloned into the linearized pTK1A-TPA vector by enzymatic restriction. Neoantigen-based vaccines are DNA plasmids encoding for neoepitopes selected from a murine colon adenocarcinoma cell line (i.e., MC38 cell line, ATCC, USA). Once identified by RNA sequencing, genes encoding for selected neoepitopes were cloned in pTK1A vector, as previously described. [7 (link), 18 (link)]
conTRT vaccine consisted of a DNA plasmid encoding a consensus sequence between canine and human telomerase. In addition, this catalytically inactive telomerase protein has been fused to a TPA (human tissue plasminogen activator) leader sequence at N-term and fused itself to the Profilin-like sequence of Toxoplasma gondii (PFTG) at the C-terminus (TPA-conTRT-PFTGopt). This construct was finally cloned into the linearized pTK1A-TPA vector by enzymatic restriction. Neoantigen-based vaccines are DNA plasmids encoding for neoepitopes selected from a murine colon adenocarcinoma cell line (i.e., MC38 cell line, ATCC, USA). Once identified by RNA sequencing, genes encoding for selected neoepitopes were cloned in pTK1A vector, as previously described. [7 (link), 18 (link)]
3
Isolation and Culture of Human Umbilical Vein Endothelial Cells
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Human umbilical vein endothelial cells (HUVECs) were isolated from normal female human umbilical veins, which were collected from Qilu Hospital of Shandong University. HUVECs were cultured in Endothelial Cell Medium (ECM; ScienCell) supplemented with 5% heat-inactivated fetal bovine serum (ScienCell), 1% penicillin/streptomycin (ScienCell), and 1% endothelial cell growth supplement (ECGS; ScienCell). All experimental primary endothelial cells were used by passage 6. HEK293T cell line (CRL-11268), LLC cell line, B16 cell line, and MC38 cell line were purchased from ATCC. HEK293T, LLC, B16, MC38 cells were cultured in DMEM/high-glucose medium supplemented with 10% fetal bovine serum and antibiotics. All cells were cultured at 37 °C and 5% CO2.
4
Pegylated Lipid-Polymer Nanoparticle Protocol
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MAL-PEG3000-b-PLA2000 was purchased from JenKem Technology Co. Ltd. All DNA sequences, fluorophore-modified CpG sequences, and thiol-modified primer were synthesized in Integrated DNA Technologies. T4 DNA ligase (4000 U/μl), phi29 DNA polymerase (10 U/μl), deoxynucleotide triphosphate (dNTP) solution mix (10 mM each nt), and 10× bovine serum albumin (BSA) in buffer solution were all purchased from New England Biolabs, and Cy5-modified dUTP was purchased from Enzo Life Sciences. R848 was purchased from Sigma-Aldrich. aPD-1 antibody was purchased from Bio X Cell Inc. All the enzyme-linked immunosorbent assay (ELISA) kits used in this study were purchased from Thermo Fisher Scientific. Staining antibodies including anti–CD8-APC-Cy7, PD1-BV421, F4/80-PE (phycoerythrin), and CD11c-APC were purchased from BioLegend. Anti–CD80-PerCP-Cy5.5 and anti–CD86-PE were purchased from PeproTech. Anti-mouse SIINFEKL/H-2Kb-PE antibody was purchased from eBioscience. Dextramer-PE was purchased from Immudex. MC38 cell line was purchased from ATCC and cultured in Dulbecco’s modified Eagle’s medium, and DC2.4 cells were cultured in RPMI 1640 medium with 2 mM l -glutamine.
5
Murine Cancer Cell Line Cultivation
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The murine melanoma B16-OVA and colon carcinoma MC38 cell line were purchased from the ATCC. Cells were cultured in RPMI 1640 (Gibco) containing 10% fetal bovine serum (FBS, Sigma-Aldrich), 2 mM glutamine (Gln, Gibco), 100 U/mL penicillin and 100 µg/mL streptomycin (100 U/mL), and 50 µM 2-mercaptoethanol (Gibco). The B16-OVA cell line was supplemented with 400 µg/mL Geneticin (Gibco). The TS/A mouse breast carcinoma cell line and its clones were kindly gifted by Dr Lorenzo Galuzzi (Weill Cornell Medical College, New York, New York, USA). All clones were cultured in DMEM (Gibco) containing 4.5 g/L glucose, 4 mM L-glutamine and 110 mg/L sodium pyruvate, supplemented with 100 mM HEPES buffer and 10% FBS (complete culture medium), 2 mM glutamine (Gln, Gibco), 100 U/mL penicillin and 100 µg/mL streptomycin (100 U/mL), and 50 µM 2-mercaptoethanol. Cell lines were routinely tested every 2 months for mycoplasma contamination (MycoAlert Mycoplasma Detection Kit, Lonza).
6
Insulin Resistance Model for Colorectal Cancer
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The human colorectal cell lines HCT116, SW480, SW620, and SW1116 were obtained from the Chinese Academy of Sciences cell bank. MC38 cell line was purchased from ATCC (CL0203). These cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin at 37 °C in 5% CO2. The insulin resistance (IR) model of CRC cells was cultured with RPMI-1640 medium containing 50mM concentration glucose for 24 h. A Glucose consumption assay was used to verify the establishment of IR. CRC cell lines (1*105 cells/mL) were seeded in 96-well plates and treated with high glucose. A glucose assay kit was supplied from Nanjing Jiancheng Bioengineering Institute (Nanjing, China), which was used to measure glucose concentrations in the culture medium. The culture medium was collected, and glucose reagent was added and then placed at 37 °C for 15 min. The OD values were measured at 505 nm. The results were calculated as follows: Glucose consumption = glucose concentration in the blank group (no cells, only medium) – glucose concentration in each group (model and control groups)[27 (link)]. The relative levels of glucose consumption in cells were normalized to that of the control group.
7
Murine Tumor Cell Lines and Antibody Depletion
8
Cell Culture Protocols for Cancer Research
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The human embryonal kidney (HEK) 293T cell line and the mouse colorectal carcinoma (CRC) MC38 cell line were obtained from the American Type Culture Collection (ATCC, Molsheim Cedex, France) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS (Harlan, Horst, The Netherlands), 2 mmol/L L-Glutamine (Sigma-Aldrich, Zwijndrecht, Belgium), 100 U/mL penicillin, 100 μg/L streptomycin (PS; Sigma-Aldrich, Zwijndrecht, Belgium).
The mouse lung carcinoma cell line TC-1 was provided by T.C. Wu (John Hopkins University, Baltimore, MD, USA) and cultured in RPMI 1640 medium supplemented with 10% FCI (Harlan, Horst, The Netherlands), 2 mmol/L L-Glutamine (L-Glu; Sigma-Aldrich, Zwijndrecht, Belgium), 100 U/mL penicillin and 100 μg/ml streptomycin (PS; Sigma-Aldrich, Zwijndrecht, Belgium), 1 mmol/L sodium pyruvate and non-essential amino acids (Sigma-Aldrich, Zwijndrecht, Belgium), 1 mM G418 (Thermofisher Scientific, Asse, Belgium), 5 mM HEPES (Thermofisher Scientific) and 50 μM beta-mercaptoethanol (Thermofisher Scientific).
The cells were cultured at 37°C 5% CO2.
The mouse lung carcinoma cell line TC-1 was provided by T.C. Wu (John Hopkins University, Baltimore, MD, USA) and cultured in RPMI 1640 medium supplemented with 10% FCI (Harlan, Horst, The Netherlands), 2 mmol/L L-Glutamine (L-Glu; Sigma-Aldrich, Zwijndrecht, Belgium), 100 U/mL penicillin and 100 μg/ml streptomycin (PS; Sigma-Aldrich, Zwijndrecht, Belgium), 1 mmol/L sodium pyruvate and non-essential amino acids (Sigma-Aldrich, Zwijndrecht, Belgium), 1 mM G418 (Thermofisher Scientific, Asse, Belgium), 5 mM HEPES (Thermofisher Scientific) and 50 μM beta-mercaptoethanol (Thermofisher Scientific).
The cells were cultured at 37°C 5% CO2.
9
Culturing Murine Colon Cancer Cells
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The murine colon adenocarcinoma cell line MC-38 cell line was purchased from American Type Culture Collection (ATCC) and cultured in 1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics at 37 °C in a humidified 5% CO2 incubator.
10
Genetically Engineered Cell Lines for Cancer Research
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The MC38 cell line was obtained from Dr Maoquan Chu (Tongji University, Shanghai, China), and the other cell lines were obtained from the American Type Culture Collection (ATCC). MC38 and Lewis lung carcinoma (LLC) cells (CRL-1642) stably expressing human CD20 antigen (MC38-CD20, LLC-CD20) and B16F10 (ATCC, CRL-6475) cells expressing the human HER2 antigen (B16-HER2) were established using lentivirus transfection as previously described.17 Cell culture medium consists of Dulbecco’s Modified Eagle Medium (DMEM;Life Technologies, Gaithersburg, Maryland, USA) and extra 10% fetal bovine serum (FBS; Gibco) at 37°C and 5% CO2 (v/v).
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