Ht 29

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The HT-29 is a cell line derived from a human colorectal adenocarcinoma. It is commonly used in cell culture research and experimentation.

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HT-29 cells (7 citations) HT-29-luc2 (4 citations) McCoy’s (for HT-29) medium (2 citations)

230 protocols using ht 29

Colorectal Adenocarcinoma Cell Culture

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The human epithelial colorectal adenocarcinoma cell lines HT-29 and Caco-2 were acquired from the Kunming Cell Bank of Type Culture Collection, Chinese Academy of Sciences (Kunming, Yunan, China). HT-29 and Caco-2 cells were cultured in RPMI 1604 medium (Gibco, Grand Island, NY, USA) and Dulbecco’s modified Eagle’s medium (DMEM) (Hyclone, Logan, UT, USA), respectively. Both media contained 10% fetal bovine serum (FBS), 1 mM pyruvate and 1 × NEAA (cat #11140050, Gibco).
Differentiated HT-29 cells were cultured as described previously, with minor modifications^{20 (link)}. Briefly, glucose and pyruvate-free RPMI 1640 culture medium (cat #11879-020, Gibco) with 10% FBS was repeatedly supplemented with 20 mM glucose or 5 mM galactose for HT-29 culture. The numbers of dead cells did not significantly increase after transfer from normal RPMI 1640 medium to the indicated glucose or galactose medium. Cell-based assays were carried out after at least 5 days of cultivation. For EGF treatment, Caco-2 cells were pre-starved in serum-free medium for 10 hr and then stimulated with EGF (200 ng/ml; Millipore, Bedford, MA, USA).

Zhang C., Mao H.L, & Cao Y. (2017). Nuclear accumulation of symplekin promotes cellular proliferation and dedifferentiation in an ERK1/2-dependent manner. Scientific Reports, 7, 3769.

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Culturing and Transfecting Cell Lines for Functional Assays

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HT-29 and MDA-MB-231 cells were purchased from ATCC. MDA-MB-231 and HT-29 cells were grown in DMEM/F12 medium (Gibco, 11320082) containing 10% FBS (Gibco, 16000044). HeLa Kyoto and RPE1 acceptor cell lines were both generous gifts received from Ryoma Ohi, University of Michigan. HeLa Kyoto and RPE1 cells were cultured at 37°C with 5% CO₂ in MEM-α medium (Gibco, 12561072) containing 10% fetal bovine serum (FBS) (Gibco, 16000044). Acceptor cell lines were maintained in blasticidin (Thermo Fisher Scientific, R21001) until generation of inducible clones, after which cell lines were maintained in puromycin (Thermo Fisher Scientific, A11138-03). For siRNA transfections in a 24-well plate format, MDA-MB-231, HT-29, HeLa Kyoto, and RPE1 cells were treated with 5 pmol siRNA that was preincubated for 5–10 min in Opti-MEM Reduced-Serum Media (Gibco, 31985062) with a Lipofectamine RNAiMax transfection reagent (Invitrogen, 13778150). KIF18A siRNAs used were a 1:1 mixture of two of the following silencer or silencer select validated siRNAs (5′ to 3’ sequence): GCUGGAUUUCAUAAAGUGGtt (Ambion, AM51334), GCUUGUUCCAGAAUCGAGAtt (Ambion, 4390824), or CGUUAACUGCAGACGUAAAtt (Ambion, 4392420). Control siRNAs used were Ambion Silencer Select Negative Control #2 (Ambion, 4390846), Silencer Negative Control siRNA #2 (Ambion, AM4613), or Silencer Select Negative Control #1 (4390843).

Schutt K.L., Queen K.A., Fisher K., Budington O., Mao W., Liu W., Gu X., Xiao Y., Aswad F., Joseph J, & Stumpff J. (2024). Identification of the KIF18A alpha-4 helix as a therapeutic target for chromosomally unstable tumor cells. Frontiers in Molecular Biosciences, 11, 1328077.

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STING Agonists Modulate HT-29 Colon Cancer

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The human colon cancer line HT-29 was obtained from the American Type Culture Collection (ATCC, American). HT-29 cells were cultured in RPMI-1640 (Gibco, USA) supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco) and 1% (v/v) penicillin-streptomycin (Gibco) and maintained at 37°C and 5% CO₂STING pathway agonists DMXAA and human recombinant ANP were added to the cell culture medium of HT-29, where the concentration of DMXAA and ANP was 10 ug/ml, and the intervention time was 24 h.

Chen C., Zhang Y., Tao M., Zhao X., Feng Q., Fei X, & Fu Y. (2022). Atrial Natriuretic Peptide Attenuates Colitis via Inhibition of the cGAS-STING Pathway in Colonic Epithelial Cells. International Journal of Biological Sciences, 18(4), 1737-1754.

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Cell Culture and siRNA Transfection Protocol

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HT-29 and MDA-MB-231 cells were purchased from ATCC. MDA-MB-231 and HT-29 cells were grown in DMEM/F12 medium (Gibco, 11320082) containing 10% FBS (Gibco, 16000044). HeLa Kyoto and RPE1 acceptor cell lines were both generous gifts from Ryoma Ohi, University of Michigan. HeLa Kyoto and RPE1 cells were cultured at 37°C with 5% CO₂ in MEM-α medium (Gibco, 12561072) containing 10% Fetal Bovine Serum (FBS) (Gibco, 16000044). Acceptor cell lines were maintained in Blasticidin (Thermo Fisher Scientific, R21001) until generation of inducible clones, after which cell lines were maintained in Puromycin (Thermo Fisher Scientific, A11138-03). For siRNA transfections in a 24-well plate format, MDA-MB-231, HT-29, HeLa Kyoto and RPE1 cells were treated with 5 pmol siRNA that was preincubated for 5-10 minutes in Opti-MEM Reduced Serum Media (Gibco, 31985062) with Lipofectamine RNAiMax Transfection Reagent (Invitrogen, 13778150). KIF18A siRNAs used were a 1:1 mixture of two the following Silencer or Silencer Select Validated siRNAs (5’ to 3’ sequence): GCUGGAUUUCAUAAAGUGGtt (Ambion, AM51334), GCUUGUUCCAGAAUCGAGAtt (Ambion, 4390824) or CGUUAACUGCAGACGUAAAtt (Ambion, 4392420). Control siRNAs used were Ambion Silencer Select Negative Control #2 (Ambion, 4390846), Silencer Negative Control siRNA #2 (Ambion, AM4613), or Silencer Select Negative Control #1 (4390843).

Schutt K., Queen K.A., Fisher K., Budington O., Mao W., Liu W., Xiao Y., Aswad F., Joseph J, & Stumpff J. (2023). Identification of the KIF18A alpha-4 helix as a therapeutic target for chromosomally unstable tumor cells. bioRxiv.

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Culturing Human Colon Cancer Cell Lines

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Human colon cancer cell lines, HT29 and Caco2, were obtained from American Type Tissue Culture Collection. HT29 were grown in McCoy medium (Gibco) supplemented with heat-inactivated 10% fetal calf serum (FCS), and 100 μg/ml pen/strep (penicillin and streptomycin, Gibco, 26600080). Caco2 were cultured in EMEM medium (Lonza, BE12-611F/12) supplemented with 10% FCS, 100 μg/ml pen/strep, 1% non-essential amino acids, 1% L-glutamine and 1% sodium pyruvate (Gibco, 11360–039). All cells were cultured in a humidified 37°C/5% CO₂ incubator. Green fluorescent protein (GFP) expressing HT29 were generated by transfection with pMONO-neo-GFP plasmid containing GFP and neomycin resistance genes (Invivogen, pmonon-gfp) by electroporation (Nucleofector, Lonza, Belgium). GFP-transfected HT29 were then selected under Geneticin (Gibco, 10131035).

Yu Y., Blokhuis B., Derks Y., Kumari S., Garssen J, & Redegeld F. (2018). Human mast cells promote colon cancer growth via bidirectional crosstalk: studies in 2D and 3D coculture models. Oncoimmunology, 7(11), e1504729.

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EMT Induction in CRC Cell Lines

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Human CRC cell lines Caco-2, HT-29, Colo-320, Colo-205, and DLD-1, and a normal colonic endothelial cell line CCD-18Co were all purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). Caco-2, HT-29, and CCD-18Co cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco-BRL, Gaithersburg, MD, USA). DLD-1, HT-29, Colo-320, and Colo-205 were grown in Roswell Park Memorial Institute (RPMI; Gibco-BRL) medium. Both of the media were supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco-BRL). The cells were maintained at 37°C in a 5% CO₂ humid atmosphere.
For EMT induction, the cells were treated with transforming growth factor-β1 (TGF-β1) (10 ng/ml) for 24 h following serum starvation for 8 h.
LY294002 (20 μM) and AZD8055 (0.8 nM) both purchased from Sigma-Aldrich (St. Louis, MO, USA) were used as inhibitors of the PI3K/AKT and mTOR pathways, respectively.

Shuai F., Wang B, & Dong S. (2018). MicroRNA-204 Inhibits the Growth and Motility of Colorectal Cancer Cells by Downregulation of CXCL8. Oncology Research, 26(8), 1295-1305.

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Comprehensive Cancer Cell Line Characterization

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CRC cell lines HCT-116 (obtained 2018), HT-29 (obtained 2018), and LoVo (obtained 2018), lung cancer cell line HOP-92 (obtained 2017), breast cancer cell line MCF7 (obtained 2021) and melanoma cell line RPMI-7951 (obtained 2021) were obtained through the NCI Cell Line Repository (Division of Cancer Treatment and Diagnosis (DCTD) Tumor Repository, NCI at Frederick, Frederick, MD). The prostate cancer cell line PC-3 were provided by Dr. Esta Sterneck (NCI-Frederick, obtained 2019). HCT-116, HT-29, LoVo, HOP-92, MCF7, RPMI-7951, and PC-3 cells were cultured in RPMI 1640 Medium supplemented with 10% FBS, 1% P/S, and 2mM L-Glutamine. All cells were incubated at 5% CO₂ at 37⁰C. Cell lines were tested for mycoplasma contamination by PPLO culture under aerobic and anaerobic conditions and orcein staining of Fogh or by PCR-based assay. Cell lines obtained from the NCI DCTD Tumor Respository (HCT-116, HT-29, LoVo, HOP-92, MCF7, RPMI-7951) were authenticated using Applied Biosystems AmpFISTR^® Identifiler with PCR amplification prior to cell line receipt. PC-3 cells were authenticated using CellCheck, (IDEXX BioAnalytics, Columbia, MO) a comprehensive cell line authentication service that utilizes STR-based DNA profiling and multiplex PCR to detect both contamination and misidentification of cell lines.

Loomans-Kropp H.A., Song Y., Gala M., Parikh A.R., Van Seventer E.E., Alvarez R., Hitchins M.P., Shoemaker R.H, & Umar A. (2022). Methylated Septin9 (mSEPT9): A Promising Blood-Based Biomarker for the Detection and Screening of Early-Onset Colorectal Cancer. Cancer Research Communications, 2(2), 90-98.

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Authenticated Cell Lines for Cancer Research

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Colorectal cancer cell lines HCT-116 (obtained 2018), HT-29 (obtained 2018), and LoVo (obtained 2018), lung cancer cell line HOP-92 (obtained 2017), breast cancer cell line MCF7 (obtained 2021), and melanoma cell line RPMI-7951 (obtained 2021) were obtained through the NCI Cell Line Repository [Division of Cancer Treatment and Diagnosis (DCTD) Tumor Repository, NCI at Frederick, Frederick, MD]. The prostate cancer cell line PC-3 was provided by Dr. Esta Sterneck (NCI-Frederick, obtained 2019). HCT-116, HT-29, LoVo, HOP-92, MCF7, RPMI-7951, and PC-3 cells were cultured in RPMI1640 medium supplemented with 10% FBS, 1% penicillin/streptomycin, and 2 mmol/L l-glutamine. All cells were incubated at 5% CO₂ at 37°C. Cell lines were tested for Mycoplasma contamination by PPLO culture under aerobic and anaerobic conditions and orcein staining of Fogh or by PCR-based assay. Cell lines obtained from the NCI DCTD Tumor Repository (HCT-116, HT-29, LoVo, HOP-92, MCF7, RPMI-7951) were authenticated using Applied Biosystems AmpFISTR Identifiler with PCR amplification prior to cell line receipt. PC-3 cells were authenticated using CellCheck (IDEXX BioAnalytics), a comprehensive cell line authentication service that utilizes STR-based DNA profiling and multiplex PCR to detect both contamination and misidentification of cell lines.

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Cell Line Culture Protocols for Research

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All the common cell lines including THP‐1 (ATCC TIB‐202), HT‐29 (ATCC HTB‐38), and HEK293T (ATCC CRL‐3216) cells were obtained from the American Type Cell Culture (ATCC), US. HEK‐Blue hNOD2 cells were purchased from InvivoGen and maintained as per the instructions. HT‐29, HEK293T, and HeLa cells were maintained in DMEM (GIBCO) supplemented with 10% heat‐inactivated fetal bovine serum (FBS) and penicillin/streptomycin. THP‐1 cells were maintained in RPMI‐1640 (GIBCO) supplemented with 10% heat‐inactivated FBS, Glucose (5%), HEPES buffer (10 mM), L‐glutamine (5 mM), sodium pyruvate (1 mM), and penicillin/streptomycin. All the cell lines were tested for mycoplasma contamination routinely (every 2–3 months). All the experiments were performed with cells below the 20^th passage.

Mehto S., Jena K.K., Yadav R., Priyadarsini S., Samal P., Krishna S., Dhar K., Jain A., Chauhan N.R., Murmu K.C., Bal R., Sahu R., Jaiswal P., Sahoo B.S., Patnaik S., Kufer T.A., Rusten T.E., Chauhan S., Prasad P, & Chauhan S. (2022). Selective autophagy of RIPosomes maintains innate immune homeostasis during bacterial infection. The EMBO Journal, 41(23), e111289.

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Establishing CRC Cell Lines and Stable Transfectants

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The human CRC cell lines HCT116 and HT29 were obtained from the Cell Bank of Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. SW480 cells were bought from ATCC. HCoEpiC and RKO cells were bought from Cobioer (Nanjing, China). HCT116 and HT29 cells were maintained in McCoy’s 5A medium supplemented with 10% fetal bovine serum (FBS); RKO and SW480 cells were cultured in 1640 medium (Gibco, 11875–093) containing 10% FBS; and HCoEpiC cells were cultured in minimum essential medium (MEM; Gibco, 11095–080) containing 10% FBS. All cells were grown in a 5% CO₂ cell culture incubator at 37°C. For stable cell line construction, HCT116 and RKO cells were transfected with plasmids using PolyJet™ DNA In Vitro Transfection Reagent (SignaGen Laboratories, SL100688). After 48 h, cells were subjected to selection with puromycin (4–6 μg/mL; J593, Amresco Inc) or G418 (1000–1500 μg/mL; sc-29065, Dallas, TX, USA) according to the antibiotic resistance of different transfected plasmids.

Wang C., Li H., Wu L., Jiao X., Jin Z., Zhu Y., Fang Z., Zhang X., Huang H, & Zhao L. (2021). Coiled-Coil Domain-Containing 68 Downregulation Promotes Colorectal Cancer Cell Growth by Inhibiting ITCH-Mediated CDK4 Degradation. Frontiers in Oncology, 11, 668743.

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