HAdV-F41 (ATCC® VR-930™) was grown in 50–60% confluent HEK-293 cells (ATCC® CRL-1573™) in DMEM (ATCC® 30-2002) supplemented with 1–2% FBS (ATCC® 30-2020™). Infection was done with virus at passage five at an MOI = 1. After infection, when cells show clear cytopathic effect (round up with increased nucleus size), cultures were harvested with a cell scraper and transferred to falcon tubes. Cell suspensions were centrifuged at 700× g, 4 °C for 10 min, and cells were resuspended in culture medium discharging the supernatant. Samples were subjected to three freeze/thaw cycles (−80 °C and 37 °C), then centrifuged at 1500× g, 4 °C for 10 min. Supernatants were aliquoted in small volumes and kept at −80 °C until use. To determine viral titers, an aliquot of the virus preparation was used for titration in HEK-293 cells via immunohistochemistry using the QuickTiter™ Adenovirus Quantitation Kit (Cell Biolabs, Catalog no. VPK-109, San Diego, CA, USA), following instructions by the manufacturer. Two cell types were used for the in vitro infection models: human colorectal carcinoma HCT116 cells (ATCC® CCL-247, Manassas, VI, USA). HCT116 cells were grown in McCoy’s 5A medium (ATCC® 30-2007™, Manassas, VI, USA) supplemented with 10% FBS.
Hct116 cell
Manufactured by American Type Culture Collection
Sourced in United States, China
HCT116 cells are a widely used human colorectal carcinoma cell line. They are adherent cells that can be used for various cell biology and cancer research applications.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (35 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (16 mentions)
Streptomycin, by Thermo Fisher Scientific (12 mentions)
Hela cell, by American Type Culture Collection (11 mentions)
Penicillin, by Thermo Fisher Scientific (11 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (10 mentions)
Mccoy s 5a medium, by Thermo Fisher Scientific (9 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (8 mentions)
Mccoy s 5a media, by Thermo Fisher Scientific (8 mentions)
Mcf 7 cell, by American Type Culture Collection (7 mentions)
Glutamax, by Thermo Fisher Scientific (7 mentions)
Hek293 cell, by American Type Culture Collection (6 mentions)
Ht 29 cell, by American Type Culture Collection (6 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (6 mentions)
A549 cell, by American Type Culture Collection (6 mentions)
Sw480 cells, by American Type Culture Collection (5 mentions)
Hepg2 cell, by American Type Culture Collection (5 mentions)
Mccoy s 5a modified medium, by Thermo Fisher Scientific (5 mentions)
Hct116, by Thermo Fisher Scientific (5 mentions)
Huvecs, by Lonza (4 mentions)
175 protocols using hct116 cell
1
Adenovirus Propagation and Titration
2
Comparative Analysis of CD44 Expression
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Human colorectal carcinoma HCT116 cells and mouse embryonic fibroblast NIH3T3 cells were purchased from ATCC (Manassas, VA, USA). HCT116 cells were maintained in McCoy's 5A modified medium (ATCC) with 10% fetal bovine serum (FBS, HyClone; Thermo Fisher Scientific K.K., Waltham, MA, USA), penicillin (100 units/mL) and streptomycin (100 μg). NIH3T3 cells were maintained in Dulbecco's modified Eagle medium (Invitrogen) with 10% FBS, penicillin (100 units/mL) and streptomycin (100 μg). These cells were cultured under an atmosphere of 5% CO2/air at 37 o C. In this experiment, an HCT116 cell line and an NIH3T3 cell line were used as a CD44-positive and CD44-negative cells, respectively.
3
Cell Culture Protocol for CMT93 and HCT116
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CMT93 cells (purchased from ATCC, Manassas, VA, USA) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (Life Technologies, Grand Island, NY, USA) and 1% penicillin G/streptomycin. HCT116 cells (purchased from ATCC, Manassas, VA, USA) were cultured in McCoy's 5A (modified) medium supplemented with 10% fetal bovine serum (Life Technologies, Grand Island, NY, USA) and 1% penicillin G/streptomycin. Cells were maintained in a CO2 AutoZero incubator (Thermo, Massachusetts, USA) at 37°C with 5% CO2.
4
Cell Culture Maintenance and Characterization
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HCT116 cells (ATCC, Manassas, VA) were maintained in McCoy’s 5A media (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (Axenia Biologix, Dixon, CA), 100 units/mL penicillin, and 100 ug/mL streptomycin (Gibco). Mutant HCT116 cells were obtained from Dominic Hoepfner (Krastel et al., 2015 (link)). HEK293T cells (ATCC) and HeLa cells (ATCC) were maintained in DMEM (Gibco) supplemented with 10% fetal bovine serum, 100 units/mL penicillin, and 100 μg/mL streptomycin. All other cell lines were obtained from the Cancer Cell Line Encyclopedia collection and were maintained as described (Barretina et al., 2012 (link)). All cell lines were tested for mycoplasma (Lonza, CH) before use and were SNP-typed to verify that their identity matched that of the originally procured sample. None of the tested lines are present in the list of cross-contaminated/misidentified cell lines (http://iclac.org/wp-content/uploads/Cross-Contaminations-v7_2.pdf ). All cells were cultured at 37°C in a 5% CO2 atmosphere.
5
Culturing Primary Epithelial Cells and Organoids
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Primary human colon epithelial cells (HCEC-1CT), a generous gift from Dr. Jerry W. Shay, were cultured as described previously [14] (link). HCT116 cells were purchased from ATCC. Small intestinal organoids (SIO) were isolated, from C57BL/6 wild type (WT) and PAK1−/− (PAK1 KO) mice (purchased from MMRRC UNC, Chapel Hills, NC), and maintained as previously described [15] (link). All cells were serum starved in incomplete medium for 12 h prior to treatment with recombinant protein. Reagents (Supplementary Table S2) were applied 2–12 h prior to inflammatory cytokines. Cells were harvested at 60–70% confluency.
6
HCT-116 Colorectal Cancer Cell Apoptosis
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HCT-116 cells, a human colorectal carcinoma cell line (ATCC, Rockville, MD), were maintained in a CO2 incubator at 37°C under a humidified atmosphere (95% air, 5% CO2) in RPMI 1640 medium supplemented with 10% fetal bovine serum, 25 mM HEPES buffer, and 1% Pen-Strep Cocktail (Gibco BRL, Gaithersburg, MD). Cells were treated with several concentrations of HMF (25, 50, and 100 μM) for 24 h to elucidate the underlying mechanism of apoptosis-induced cell death.
7
Generation of HCT116 CRISPRi Cell Line
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HCT116 cells (ATCC) were cultured in McCoy’s 5a media (Thermo Fisher Scientific) supplemented with 10% FBS (Gibco, Sigma-Aldrich) and 1X AA (Gibco). The cell line was authenticated by ATCC using short tandem repeat (STR) analysis in November 2017. All cell lines were confirmed to be free of mycoplasma contamination. HCT116 NORAD–/– cells were generated previously (Lee et al., 2016 (link)).
To generate the HCT116 CRISPRi cell line, lentivirus expressing dCas9/BFP/KRAB was produced by first seeding 6 × 105 HEK293T cells per well in a six-well plate. The following day, cells were transfected with 1.4 μg of pHR-SFFV-dCas9-BFP-KRAB (Addgene plasmid #46911), 0.84 μg of psPAX2 (Addgene plasmid #12260), 0.56 μg of pMD2.G (Addgene plasmid #12259), 8.4 μl of FuGENE HD (Promega), and 165 μl Opti-MEM (Thermo Fisher) according to the manufacturer’s instructions. Medium was changed the next day. Two days after transfection, medium was collected and passed through a 0.45 μm SFCA sterile filter. Recipient HCT116 cells were transduced overnight using medium supplemented with 8 μg/ml polybrene (EMD Millipore). Cells expressing BFP were enriched by FACS and single-cell clonal lines were derived.
To generate the HCT116 CRISPRi cell line, lentivirus expressing dCas9/BFP/KRAB was produced by first seeding 6 × 105 HEK293T cells per well in a six-well plate. The following day, cells were transfected with 1.4 μg of pHR-SFFV-dCas9-BFP-KRAB (Addgene plasmid #46911), 0.84 μg of psPAX2 (Addgene plasmid #12260), 0.56 μg of pMD2.G (Addgene plasmid #12259), 8.4 μl of FuGENE HD (Promega), and 165 μl Opti-MEM (Thermo Fisher) according to the manufacturer’s instructions. Medium was changed the next day. Two days after transfection, medium was collected and passed through a 0.45 μm SFCA sterile filter. Recipient HCT116 cells were transduced overnight using medium supplemented with 8 μg/ml polybrene (EMD Millipore). Cells expressing BFP were enriched by FACS and single-cell clonal lines were derived.
8
Cell Line Culture Conditions
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Cells lines in this study were grown at 37 °C with 5% CO2. Cell lines were cultured in media supplemented with 10% fetal bovine serum (Atlanta Biologicals, Flowery Branch, CA, USA, #S11150) and penicillin/streptomycin. HCT116 cells, purchased from ATCC (Manassas, VA, USA), were cultured in McCoy's 5A medium and 293GP cells were cultured in DMEM.
9
Generating 5-Fu and OXA-resistant Cells
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HCT116 cells (ATCC CCL-247; American Type Culture Collection, Manassas, VA, USA) and LS174T cells (ATCC CL-188) were cultured at 37°C in 5% CO2 atmosphere in McCoy’s 5a Medium (ATCC 30-2007) and Eagle’s Minimum Essential Medium (ATCC 30-2003), respectively with 10% FBS for less than 6 months. HCT116 and LS174T 5-Fu-resistant cells were generated by incubating with 5 µM and 10 µM 5-Fu, respectively, for 3 months. HCT116 and LS174T OXA-resistant cells were generated by incubating with 7 µM and 15 µM OXA, respectively for 3 months.
10
Cell Culture Conditions for HEK293T, HCT116, JURKAT, and OVCAR8
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HEK293T (ATCC) and HCT116 cells (ATCC) were cultured in complete media: DMEM supplemented with 10% fetal bovine serum (Invitrogen), 2 mM glutamine, 100 U/mL penicillin and 100 mg/mL streptomycin (all from Gibco). JURKAT (ATCC) and OVCAR8 (ATCC) cells were cultured in complete media: RPMI 1640 supplemented with 10% fetal bovine serum (Invitrogen), 2 mM glutamine, 100 U/mL penicillin and 100 mg/mL streptomycin (all from Gibco). Cells were maintained in a humidified incubator at 37 ˚C.
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