Caco 2 cell line

Manufactured by European Collection of Cell Cultures

Sourced in United Kingdom

The Caco-2 cell line is a human epithelial colorectal adenocarcinoma cell line commonly used in in vitro studies. The cell line consists of heterogeneous human colon carcinoma cells that exhibit morphological and functional characteristics of mature intestinal epithelial cells.

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12 protocols using caco 2 cell line

Caco-2 Cell Culture Assay for 4-AN

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Minimum Essential Medium (MEM) with Earl’s Balanced Salts (MEM/EBSS) and 2 mM l-Glutamine was obtained from HyClone Thermo Scientific (South Logan, Utah, USA). Non-essential amino acids (NEAA), fetal bovine serum (FBS), penicillin/streptomycin solution (P/S), 0.25% (w/v) trypsin – 0.53 mM EDTA, and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO, USA).
The Caco-2 cell line was obtained from the European Collection of Cell Cultures. The cells were cultured in MEM supplemented with 1% NEAA, 100 IU, 0.1 mg/ml P/S, and 10% FBS – complete MEM (cMEM) at 37 °C, 5% CO₂.
The 4-AN compound was dissolved in DMSO to obtain a stock solution. Working solutions (40, 4, 0.4 µg/ml) were prepared with the use of cMEM. The highest concentration of DMSO in the working solution did not exceed 0.1%.

Masłyk M., Janeczko M., Demchuk O.M., Boguszewska-Czubara A., Golczyk H., Sierosławska A., Rymuszka A., Martyna A, & Kubiński K. (2017). A representative of arylcyanomethylenequinone oximes effectively inhibits growth and formation of hyphae in Candida albicans and influences the activity of protein kinases in vitro. Saudi Pharmaceutical Journal : SPJ, 26(2), 244-252.

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Enterococcal Adhesion to Caco-2 Cells

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The colonocyte-like cell line Caco-2, was used to determine the adhesion ability of enterococcal strain BGGO9-28. A Caco-2 cell line was purchased from the European Collection of Cell Cultures (ECACC No. 86010202). Adhesion to the Caco-2 cell line was done according to Sánchez et al. (2010 (link)). The results of adhesion were expressed as percentage (%) (colony forming units (CFU) adhered bacteria/CFU added bacteria × 100) from two replicated plates. In each plate three wells were used per sample.

Veljović K., Popović N., Miljković M., Tolinački M., Terzić-Vidojević A, & Kojić M. (2017). Novel Aggregation Promoting Factor AggE Contributes to the Probiotic Properties of Enterococcus faecium BGGO9-28. Frontiers in Microbiology, 8, 1843.

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Caco-2 Cell Culture and Maintenance

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The Caco-2 cell line were purchased from the European Collection of Cell Cultures (Salisbury, United Kingdom—catalogue No. 8601020). Cells were cultured in Nunc™ EasyFlask™ cell culture flasks (Thermo Fisher, Darmstadt, Germany) in DMEM (containing: 0.584 g/L L-glutamine and 4.5 g/L D-glucose), supplemented with 10% (v/v) FBS, 3.7 g/L sodium hydrogencarbonate, 1% (v/v) non-essential amino acid solution, and 100 IU/mL penicillin K, with 100 μg/mL streptomycin sulfate at 37 °C in an atmosphere of 5% CO₂. Passaging was regularly performed for cell maintenance and fresh glutamine was continuously supplemented by GlutaMax™. Our experiments were carried out on cultures between passage numbers 25–40 [14 (link)].

Pham Le Khanh H., Nemes D., Rusznyák Á., Ujhelyi Z., Fehér P., Fenyvesi F., Váradi J., Vecsernyés M, & Bácskay I. (2022). Comparative Investigation of Cellular Effects of Polyethylene Glycol (PEG) Derivatives. Polymers, 14(2), 279.

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Caco-2 Cell Culture and Maintenance

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Caco-2 cell line was purchased from the European Collection of Cell Cultures (ECACC No. 86010202). The culture and maintenance of the cell line was carried out as previously described [16 (link)] using Dulbecco’s modified Eagle’s medium (Thermo Fisher Scientific, USA) with 10% foetal bovine serum (FBS) (Thermo Fisher Scientific, USA), 100 U/ml of penicillin, 100 μg/ml of streptomycin and 4 mmol/L glutamine (Sigma, St. Louis, MO, USA). For the experiments 2 × 10⁵ Caco-2 cells per well (passage 15–30) were seeded in 24-well plates (Corning Costar, Sigma-Aldrich, Munich, Germany), cultivated at 37 °C in a 5% CO₂-humidified incubator and collected at 60% confluence.

Golić N., Veljović K., Popović N., Djokić J., Strahinić I., Mrvaljević I, & Terzić-Vidojević A. (2017). In vitro and in vivo antagonistic activity of new probiotic culture against Clostridium difficile and Clostridium perfringens. BMC Microbiology, 17, 108.

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Insulin Permeability Enhancement in Caco-2 Cells

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Human recombinant insulin, pepsin (≥400 unit/mg protein), and pancreatin (≥3× USP specifications) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Sodium alginate was purchased from BÜCHI Labortechnik AG (Flawil, Switzerland). Calcium chloride dihydrate was ordered from VWR International (Debrecen, Hungary). Labrasol ALF (Caprylocaproyl Prolyoxyl-8-glycerides) and Labrafil M2125 CS (Linoleoyl Polyoxyl-6 glycerides) were purchased from Gattefossé (Saint-Priest, France). The Caco-2 cell line was obtained from the European Collection of Cell Cultures (ECACC, Public Health England, Salisbury, UK). MTT dye (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), phosphate buffered saline (PBS) buffer solution, Dulbecco’s Modified Eagle’s Medium (DMEM), heat-inactivated fetal bovine serum (FBS), L-glutamine, non-essential amino acids solution, and penicillin-streptomycin solution were obtained from Sigma-Aldrich (St. Louis, MO, USA). TrypLE™ Express Enzyme (no phenol red) and Pierce™ Detergent Compatible Bradford Assay Kit were ordered from Thermo Fisher Scientific (Waltham, MA, USA).

Bácskay I., Papp B., Pártos P., Budai I., Pető Á., Fehér P., Ujhelyi Z, & Kósa D. (2023). Formulation and Evaluation of Insulin-Loaded Sodium-Alginate Microparticles for Oral Administration. Pharmaceutics, 16(1), 46.

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Caco-2 Cell Line Culture Protocol

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The colon adenocarcinoma (Caco-2) cell line was received from the European Collection of Cell Cultures (ECACC, Salisbury, United Kingdom, catalogue No.86010202). Caco-2 cell line is a well-established cell culture in our laboratory. As it can be seen in the article of Nemes et al. the cell culture is used in based on the same protocol. We state that the Caco-2 cell line provenance is from ECACC. Cells were seeded in plastic cell culture flasks (Thermo Fisher Scientific Inc., Budapest, Hungary in DMEM medium supplemented with 3.7 g/L NaHCO₃, 10% (v/v) heat-inactivated fetal bovine serum (FBS), 1% (v/v) non-essential amino acids solution, 1% (v/v) l-glutamine, 100 IU/mL penicillin, and 100 µg/mL streptomycin at 37 °C in an atmosphere of 5% CO₂. For the cytotoxic experiment, cells between 20–40 in passage number were used. The culture media was replaced with fresh media every three or four days [32 (link)].

Arany P., Róka E., Mollet L., Coleman A.W., Perret F., Kim B., Kovács R., Kazsoki A., Zelkó R., Gesztelyi R., Ujhelyi Z., Fehér P., Váradi J., Fenyvesi F., Vecsernyés M, & Bácskay I. (2019). Fused Deposition Modeling 3D Printing: Test Platforms for Evaluating Post-Fabrication Chemical Modifications and In-Vitro Biological Properties. Pharmaceutics, 11(6), 277.

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Caco-2 Cell Line Characterization

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The Caco-2 cell line was from the European Collection of Cell Cultures (ECACC, number 86,010,202, Salisbury, UK). Tests were performed between passages 26 and 31. The culture medium was EMEM (Eagle Minimum Essential Medium) with phenol red, to which 10% fetal bovine serum, 1% glutamine, 1% nonessential amino acids, and 1% antibiotic (penicillin and streptomycin) were added. The cell line was maintained in 7.5 cm² flasks, changing the medium every two days, and their growth was observed by inverted phase contrast microscopy. The flasks were kept at 37 °C with 5% CO₂ at 95% humidity. A subculture was performed when the cell culture reached 80% confluence.
The cell monolayer viability was measured with an MTT assay [24 (link)] and a mycoplasma test to verify the purity of the cell line was performed. On the other hand, the cell monolayer integrity was evaluated by measuring the transepithelial electrical resistance (TEER) while using an apparent permeability test (phenol red test) [25 ].

Martínez L., Ros G, & Nieto G. (2018). Fe, Zn and Se Bioavailability in Chicken Meat Emulsions Enriched with Minerals, Hydroxytyrosol and Extra Virgin Olive Oil as Measured by Caco-2 Cell Model. Nutrients, 10(8), 969.

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Caco-2 Cell Culture Conditions

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Caco-2 cell line was obtained from the European Collection of Cell Cultures (ECACC, Salisbury, United Kingdom). Cells were grown in plastic cell culture flasks in Dulbecco’s Modified Eagle’s Medium, supplemented with 3.7 g/L NaHCO₃, 10% (v/v) heat-inactivated fetal bovine serum (FBS), 1% (v/v) non-essential amino acids solution, 1% (v/v) L-glutamine, 100 IU/mL penicillin, and 100 µg/mL streptomycin at 37 °C in an atmosphere of 5% CO₂. The cells were routinely maintained by regular passaging and glutamine was supplemented by GlutaMax™. The cells used for cytotoxic experiments were between passage numbers 20 and 40.

Nemes D., Kovács R., Nagy F., Mező M., Poczok N., Ujhelyi Z., Pető Á., Fehér P., Fenyvesi F., Váradi J., Vecsernyés M, & Bácskay I. (2018). Interaction between Different Pharmaceutical Excipients in Liquid Dosage Forms—Assessment of Cytotoxicity and Antimicrobial Activity. Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry, 23(7), 1827.

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Caco-2 Cell Culture and Acute Arsenic Exposure

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The human colon Caco-2 cell line was obtained from the European Collection of Cell Cultures (ECACC, number 86010202, Salisbury, UK). Cells were grown at 37 ºC in an atmosphere with 95% relative humidity and a CO 2 flow of 5% in 75 mL flasks with 10 mL of DMEM with high glucose (4.5 g/L) and L-glutamine (0.6 g/L) supplemented with 10% (v/v) of FBS, 1% (v/v) of nonessential amino acids, 1 mM of sodium pyruvate, 10 mM of HEPES, 100 U/mL of penicillin, 0.1 mg/mL of streptomycin and 0.0025 mg/L of amphotericin B (DMEM-10%FBS). The medium was changed every 2-3 days. When the cell monolayer reached 80% confluence, the cells were detached with a solution consisting of 0.5 g/L of trypsin and 0.22 g/L of ethylene diamine tetraacetic acid (EDTA), and reseeded at a density of 5 × 10 4 cells/cm 2 . All the reagents used for the cell maintenance were obtained from Hyclone. The assays were performed with cultures between passages 5 to 25.
For As acute exposure (sections 2.4-2.5), Caco-2 cells were seeded in DMEM-10%FBS and 7 days post-seeding, subjected to the following treatments for 24 h: The selected conditions for the acute exposure were those that had the greatest toxic effect in previous experiments [16] (link).

de Matuoka E Chiocchetti G., Monedero V., Zúñiga M., Vélez D, & Devesa V. (2020). In Vitro Evaluation of the Protective Role of Lactobacillus StrainsAgainst Inorganic Arsenic Toxicity. Probiotics and antimicrobial proteins, 12(4).

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Nanoemulsion Formulation and Cell Lines

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Corn oil (Mazola, ACH Food Companies Inc., Memphis, TN) was purchased from a local market. β-carotene (synthetic, ≥93% (UV), powder) was sourced from Sigma-Aldrich (Ireland).
Lecithin was obtained from Alfa Aesar (Karlsruhe, Germany). Sodium caseinate (NaCas) (≥92% purity) was from Acros Organics (Geel, Belgium). The Caco-2 cell line was purchased from the European Collection of Cell Cultures (ECACC 86010202) and the human monocyte THP-1 (ATCCTIB-202) and the human colon adenocarcinoma HT-29 cell lines (ATCCHTB-38) were purchased from American Type Culture Collection. This latter cell line was differentiated to HT-29-MTX following the protocol described by Guri et al. (2013) . Tissue culture plastics were sourced from Sarstedt Ltd. (Wexford, Ireland). CellTiter 96 AQueus One Solution reagent was purchased from Promega (MyBio, Kilkenny, Ireland). Milli-Q water was used to prepare all nanoemulsions. All other chemicals were sourced from Sigma-Aldrich (Ireland) unless specified otherwise.

Gasa-Falcon A., Arranz E., Odriozola‐Serrano I., Martín‐Belloso O., & Giblin L. (2021). Delivery of β-carotene to the in vitro intestinal barrier using nanoemulsions with lecithin or sodium caseinate as emulsifiers. LWT, 135, 110059-110059.

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