Dld 1 ccl 221

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The DLD-1 (CCL-221) is a cell line originating from a human colon adenocarcinoma. It is a well-established tool for research purposes.

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DLD-1 (873 citations) CCL-1 (38 citations) CCL-221 (19 citations) CCL-1TM (6 citations) DLD-1 (ATCC® CCL-221™) (6 citations)

13 protocols using dld 1 ccl 221

Establishing Colorectal Cancer Cell Lines

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Human CRC specimen samples were procured from the Surgical Pathology Unit at the Chinese Academy of Medical Sciences Cancer Hospital (Beijing, China). Informed consent was obtained from all subjects, and this study was approved by the ethical committees of the Cancer Hospital, Chinese Academy of Medical Sciences. The clinical backgrounds for these patients are shown in Supplementary Tables S1–S3. Peripheral blood was collected from patients and centrifuged with Ficoll density gradient, following which the middle layer cells were gathered for fluorescence-activated cell sorting (FACS) analysis. Mice were housed at our Institutional Animal Care unit. All animal experiment protocols were approved by the ethical committee of the Chinese Academy of Medical Sciences, Cancer Hospital.
THP-1 (TIB-202, RRID:CVCL_0006), HCT116 (CCL-247, RRID:CVCL_0291) and DLD1 (CCL-221, RRID:CVCL_0248) cells were from ATCC. Ana-1 (EP-CL-0023, RRID:CVCL_0142) and CT26 cells (EP-CL-0071, RRID:CVCL_7254) were from Elabscience (Texas, USA). All the cells were maintained in RPMI 1640 (Bioroc™, China), supplemented with 10% heated-inactivated fetal bovine serum, 100 UI/mL penicillin, and 100 μg/mL streptomycin.

Zhao Y., Zhang W., Huo M., Wang P., Liu X., Wang Y., Li Y., Zhou Z., Xu N, & Zhu H. (2021). XBP1 regulates the protumoral function of tumor-associated macrophages in human colorectal cancer. Signal Transduction and Targeted Therapy, 6, 357.

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Neoadjuvant Radiotherapy in Rectal Cancer

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Patients with clinical T3 rectal cancer (patients characteristics, online supplementary table 1) received neoadjuvant hyperfractionated radiotherapy over the course of 5 days within the frame of a controlled clinical study.34 (link) As a control group, we used historical surgical tumor specimen of 25 non-pretreated rectal cancer lesions of patients from the same institution with no history of irradiation therapy or cytoablative treatment. For immunofluorescence and immunohistochemical stainings, surgical paraffin-embedded specimen of the cancer lesions were cut in 5 µm sections and mounted on slides. For ex vivo cultures and subsequent flow cytometric stainings, rectal cancer tissue was obtained from 10 patients with histologically verified rectal cancer with no history of irradiation therapy or cytoablative treatment. Studies involving patient material were performed according to the Declaration of Helsinki and approved by the local ethics committee of the Medical University of Vienna (1374/2014). For OTA, HCT116 (CCL-147) and DLD-1 (CCL-221) were purchased from ATCC. Peripheral blood mononuclear cells for macrophage differentiation were isolated from healthy blood donors.

Stary V., Wolf B., Unterleuthner D., List J., Talic M., Laengle J., Beer A., Strobl J., Stary G., Dolznig H, & Bergmann M. (2020). Short-course radiotherapy promotes pro-inflammatory macrophages via extracellular vesicles in human rectal cancer. Journal for Immunotherapy of Cancer, 8(2), e000667.

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Human Cell Line Origins and Maintenance

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Human Telomerase immortalized cell lines hTERT-RPE1 (X4000-1 46,XX retinal pigment epithelia) and hTERT-BJ1 (C4001-1 46,XY foreskin fibroblast) were originally obtained from Clontech, but are now available from the American Type Culture Collection (ATCC). Human embryonic Stem Cell lines H9 (WA09, 46,XX) and H1 (WA01, 46,XY) were obtained from WiCell Research Institute. The following human cells lines were obtained from ATCC: primary skin fibroblast cells CCD-1139Sk (CRL-2708, 46,XY) and CCD-1140Sk (CRL-2714, 46,XY); male colorectal adenocarcinoma cell lines DLD-1 [CCL-221] and HCT116 (CCL-247); fetal lung fibroblasts IMR-90 (CCL186, 46,XX) and WI-38 (CCL-75, 46,XX). Fetal human dermal fibroblasts were obtained from ScienCell Research Laboratories (Catalog Number 2300). All cells were maintained according to the supplier recommendations.

Figueroa D.M., Darrow E.M, & Chadwick B.P. (2015). Two novel DXZ4-associated long noncoding RNAs show developmental changes in expression coincident with heterochromatin formation at the human (Homo sapiens) macrosatellite repeat. Chromosome research : an international journal on the molecular, supramolecular and evolutionary aspects of chromosome biology, 23(4), 733-752.

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Colorectal Cancer Cell Line Cultivation

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Human CRC cell lines, HT29 (HTB-38), HCT116 (CCL-247), DLD1 (CCL-221), and mouse CRC cell line CT26 (CRL-2638) were purchased from ATCC in 2016. Mouse CRC cell line MC38 (ENH204-FP) was purchased from Kerafast Inc. (Boston, MA) in 2017. All the CRCs were cultured and expanded in either RMPI 1640 or DMEM (Gibco) medium supplemented with 10% heat-inactivated FBS, penicillin (100 U/mL), streptomycin (100 μg/mL), and l-glutamine (2 mmol/L) according to supplier protocols and aliquoted at low passage rates (<10) for utilization across experiments. Cells were not reauthenticated. Mycoplasma testing was performed by the Tissue Culture Support Center at Washington University School of Medicine. Epacadostat was obtained under Material Transfer Agreement from Incyte, Inc (Wilmington, DE), stored at 4 °C and was reconstituted in DMSO at 50 mM and added to cell cultures at the experimentally described concentrations. L-kynurenine (Sigma, K8625–100mg) was reconstituted in H₂O at 50 mM. IFNγ treatment (2 ng/mL) was used as described experimentally. Reagents and vendors are detailed in Supplementary Table S2.

Chen B., Alvarado D.M., Iticovici M., Kau N.S., Park H., Parikh P.J., Thotala D, & Ciorba M.A. (2020). Interferon-induced IDO1 mediates radiation resistance and is a therapeutic target in colorectal cancer. Cancer immunology research, 8(4), 451-464.

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Culturing Colorectal Cancer Cell Lines

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Human CRC cells, DLD-1 (CCL-221), HT-29 (HTB-38), and HCT116 (CCL-247), were obtained from American Type Culture Collection (ATCC, Rockville, MD, USA). All cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) (SAFC Biosciences, Lenexa, KS, USA) and 1% penicillin/streptomycin containing 100 IU/mL of penicillin and 100 μg/mL of streptomycin at 37 °C in 5% CO₂ in a humidified incubator. TTK inhibitor AZ3146 (Selleck, Houston, TX, USA) was dissolved in DMSO at a concentration of 100 mM and diluted sequentially into 4 mM, 2 mM, and 1.5 mM with DMEM containing 10% FBS.

Lee C.C., Tsai K.Y., Lee A.W., Wei P.L., Huang C.Y., Batzorig U, & Chang Y.J. (2023). CWH43 Is a Novel Tumor Suppressor Gene with Negative Regulation of TTK in Colorectal Cancer. International Journal of Molecular Sciences, 24(20), 15262.

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Cell Culture Protocol for Cancer Research

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C6-HRE-GFP cells were a gift from Dr Inna Serganova (Memorial Sloan Kettering Cancer Center, New York, NY, USA). MMTV-PyMT cells were derived by Daniela Quail (McGill University, Montreal, QC, Canada). MDA-MB-231 (HTB-26), HEK293T, hTERT RPE-1 (CRL-4000) and DLD-1 (CCL-221) cells were purchased from American Type Culture Collection. All our cells are routinely tested for contamination and authenticated. All cells except RPE-1 were grown in high-glucose Dulbecco's modified Eagle medium (DMEM; Gibco, 11965-092) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich, F0926). RPE-1 cells were grown in high-glucose DMEM-F12 medium (Gibco, 11039) containing 10% FBS. All cells were incubated at 5% CO₂ and 37°C.

Janská L., Anandi L., Kirchberger N.C., Marinkovic Z.S., Schachtner L.T., Guzelsoy G, & Carmona-Fontaine C. (2021). The MEMIC is an ex vivo system to model the complexity of the tumor microenvironment. Disease Models & Mechanisms, 14(8), dmm048942.

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Cell Culture Protocol for HEK293T, DLD-1, and G402

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Human embryonic kidney (HEK) 293T (CRL-3216), DLD-1 (CCL-221), and G402 (CRL-1440) cells were obtained from American Type Culture Collection. SYO-1 cells (39) were a gift from C. Kadoch. Cells were cultured in Dulbecco's modified Eagle's medium (Thermo Fisher Scientific, 11965-118) supplemented with 15% fetal bovine serum (MilliporeSigma, F2442), 1× Gluta-MAX (Thermo Fisher Scientific, 35050-079), and penicillin-streptomycin (100 U/ml; Thermo Fisher Scientific, 15140-163). For selection of lentivirus-infected cells, puromycin was used at a concentration of 1.5 μg/ml, and hygromycin was used at a concentration of 250 μg/ml. Cells were tested for mycoplasma contamination using a MycoStrip detection kit (Invivogen, rep-mys-100).

Morgan M.A.J., Mohammad Parast S., Iwanaszko M., Aoi Y., Yoo D., Dumar Z.J., Howard B.C., Helmin K.A., Liu Q., Thakur W.R., Zeidner J.M., Singer B.D., Eichler E.E, & Shilatifard A. (2023). ELOA3: A primate-specific RNA polymerase II elongation factor encoded by a tandem repeat gene cluster. Science advances, 9(47).

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Colorectal Adenocarcinoma Cell Lines

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DLD-1 (CCL-221) and Ht-29 (HTB-38), human colorectal adenocarcinoma cell lines, were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The results of our previous research [21 (link)] and data from the literature [22 (link)] indicate that the DLD-1 line contains the EpoR gene and protein, and histologically is the most similar to a primary tumor. Line Ht-29, in turn, is a negative control of the EpoR gene and is used to assess multidrug resistance, absorption of nutrients, and chemically induced differentiation of enterocytes [22 (link)]. Cell line DLD-1 was cultured in RPMI 1640 medium (Sigma, USA), line Ht-29 in McCoy’s 5a medium (Sigma, USA) supplemented with 10 % fetal calf serum (Sigma, USA), penicillin (50 IU, Sigma, USA), and streptomycin (50 µg/l, Sigma, USA), in an incubator with 5 % CO₂ (normoxia), at a relative humidity of 95 %, at 37 °C (Heraeus, USA). The culture media was changed every 2 days. Cells were generally maintained in 75 cm² flasks (Sarstedt, USA). However, for the experiments, cells were plated in 100-mm dishes (Sarstedt, USA) with 10 ml of medium. The control was medium with PBS only. For all the experiments, cells in the fifth to ninth passage were used.

Tankiewicz-Kwedlo A., Hermanowicz J., Surażynski A., Rożkiewicz D., Pryczynicz A., Domaniewski T., Pawlak K., Kemona A, & Pawlak D. (2016). Erythropoietin accelerates tumor growth through increase of erythropoietin receptor (EpoR) as well as by the stimulation of angiogenesis in DLD-1 and Ht-29 xenografts. Molecular and Cellular Biochemistry, 421(1), 1-18.

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Culturing Human Colon Cancer Cells

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All culture materials were purchased from Gibco (Grand Island, NY, USA). Two human colon cancer cell line DLD-1 (CCL-221) and the human colorectal carcinoma cell line HCT-116 (CCL-247) were purchased from the American Type Culture Collection (ATCC). DLD-1 cells were cultured in RPMI 1640 medium composed of 10% fetal calf serum (FCS) (S0113; Biochrom KG, Berlin, Germany) and 1% antibiotics (100 units/mL of penicillin and 100 μg/ml of streptomycin); HCT-116 cells were cultured in DMEM supplemented with 10% heat-inactivated newborn calf serum. Passage number 1 of human normal human colonic epithelial cells (HCoEpiC) was purchased from ScienCell Research Laboratories (Carlsbad, CA) and cells were grown. Both cells were maintained at 37°C in a humidified 5% CO2 incubator (Lee et al., 2013 (link)).

Lee K.C., Lee K.F., Tung S.Y., Huang W.S., Lee L.Y., Chen W.P., Chen C.C., Teng C.C., Shen C.H., Hsieh M.C, & Kuo H.C. (2019). Induction Apoptosis of Erinacine A in Human Colorectal Cancer Cells Involving the Expression of TNFR, Fas, and Fas Ligand via the JNK/p300/p50 Signaling Pathway With Histone Acetylation. Frontiers in Pharmacology, 10, 1174.

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ABCA1 Modulation in DLD-1 and Caco-2 Cells

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DLD‐1 (CCL‐221™) and Caco‐2 (HTB‐37™) were purchased from the American Type Culture Collection (ATCC^®, Manassas, VA, USA). Lentiviruses were produced in HEK293T cells. Selection was performed over 12 days by adding 2 μg·mL⁻¹ puromycin and 3 μg·mL⁻¹ blasticidin (Sigma‐Aldrich, Alcobendas, Spain) for no open reading frame (NoORF and ABCA1 constructs, respectively. Methyl‐β‐cyclodextrin (MβCD) was purchased from Sigma (Cat. C4555). Human recombinant apolipoprotein A1 (hAPOA1) (Sigma‐Aldrich; Cat. SRP.4693) was used in a final concentration of 40 μg·mL⁻¹. The absence of mycoplasma was confirmed by PCR every second week.

Aguirre‐Portolés C., Feliu J., Reglero G, & Ramírez de Molina A. (2018). ABCA1 overexpression worsens colorectal cancer prognosis by facilitating tumour growth and caveolin‐1‐dependent invasiveness, and these effects can be ameliorated using the BET inhibitor apabetalone. Molecular Oncology, 12(10), 1735-1752.

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