Caco-2 cells were obtained from China Infrastructure of Cell Line Resource (Beijing, China) and were cultured based on the method of Zhang et al. [32 (link)]. Cells were cultured in MEM containing 10% fetal bovine serum, 1 mM nonessential amino acid solution, 1% penicillin-streptomycin, and 1% HEPES buffer. Caco-2 cells were seeded at 5 × 105 cells/mL in 25 cm2 tissue culture flasks, and incubated at 37 °C in 5% CO2 with 95% humidity. The passage number of the cells used in the study was between 30 and 40. The medium in cell cultures were replaced every two days and detached from 70–80% confluency using 0.05% trypsin and 0.02% Ethylene Diamine Tetraacetic Acid (EDTA) solution. For the transport experiments, Caco-2 cells were seeded into a collagen-coated transwell plate (6.5 mm diameter, 0.4 μm pore size Corning Costar) at a density of 1 × 105 cells/well.
Caco 2 cells
Manufactured by Corning
Sourced in United States
Caco-2 cells are a human epithelial colorectal adenocarcinoma cell line derived from the colon. They are commonly used as an in vitro model for studying intestinal drug absorption and permeability.
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Lab products found in correlation
Caco 2 cell, by American Type Culture Collection (5 mentions)
Dulbecco s modified eagle s medium, by Merck Group (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Foetal bovine serum (fbs), by EQT (1 mentions)
Plastic culture dishes, by Corning (1 mentions)
Minimum essential medium (mem), by Merck Group (1 mentions)
Caco 2 cells, by Merck Group (1 mentions)
24 well plate, by Corning (1 mentions)
Caco 2 cell line, by American Type Culture Collection (1 mentions)
Potato dextrose agar (pda), by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
L glutamine, by Merck Group (1 mentions)
Sulphuric acid, by Loba Chemie (1 mentions)
Hydrochloric acid (hcl), by Loba Chemie (1 mentions)
Escherichia coli mtcc 443, by Microbial Type Culture Collection (1 mentions)
Ferric chloride anhydrous, by Loba Chemie (1 mentions)
Nitric acid, by Loba Chemie (1 mentions)
Staphylococcus aureus mtcc 3160, by Microbial Type Culture Collection (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
12 protocols using caco 2 cells
1
Caco-2 Cell Culture Protocol for Transport Experiments
2
Caco-2 Cell Culture Protocol
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Caco-2 cells were obtained from American Type Culture Collection (ATCC, Middlesex, UK) and grown in Dulbecco's Modified Eagle's medium (DMEM) supplemented with 4,500 mg/l glucose, 1% (v/v) non-essential amino acids, 10% (v/v) heat inactivated foetal bovine serum (Labtech, Sussex, UK), penicillin (100 U/ml) and streptomycin (100 U/ml) at 37 °C in 5% CO 2 and 95% humidity. Caco-2 cells were seeded at 5×10 5 cells/cm 2 into standard tissue culture coated 24 well plates (Costar, Cambridge, UK) for realtime quantitative polymerase chain reaction (RT-qPCR) analysis and cholesterol uptake assays or polycarbonate semi-permeable transwell membranes (0.4 µM pores; Costar, Cambridge, UK) for cholesterol efflux assays. Caco-2 cells were maintained for 18 to 21 days to allow complete polarisation (Natoli et al., 2012) and used when the trans-epithelial electrical resistance exceeded 900 Ω/cm 2 (Supplementary Figure S1A).
3
Caco-2 Cell Culture for Cytotoxicity Assays
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The Caco-2 cell line was obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA) and was used between passage numbers 56-62. Caco-2 cells (ATCC 37-HTB) were incubated at 37 °C in a humidified atmosphere (95% air; 5% CO 2 ) and were grown in Minimum Essential Medium (Sigma, St. Louis, MO, USA) supplemented with 15% fetal bovine serum, 25 mM HEPES, 100 units/ml penicillin, 100 μg ml -1 streptomycin and 0.25 μg ml -1 amphotericin B. Culture medium was changed every 2-3 days and the culture was split every 7 days. For subculturing, the cells were removed enzymatically (0.25% trypsin-EDTA, 5 min, 37 °C), split 1:3, and subcultured in plastic culture dishes (21 cm 2 ; ∅ 60 mm; Corning Costar, Corning, NY). For the experiments, the Caco-2 cells were seeded on 24-well plates (2 cm 2 ; ∅ 16 mm; Corning Costar). For 24 h before the experiment, the cell medium was free of fetal bovine serum. Uptake and enzymatic studies were generally performed 14 days after the cells formed a monolayer.
Caco-2 cells were treated for 24 h with CPP2 and all conjugates (1-250 μM) except for BTZ-C2-CPP2 (1-100 μM). At the end of the treatment period, proliferation, viability and assessment of cell mass studies were performed. Control cells were exposed to the respective solvent (water).
Caco-2 cells were treated for 24 h with CPP2 and all conjugates (1-250 μM) except for BTZ-C2-CPP2 (1-100 μM). At the end of the treatment period, proliferation, viability and assessment of cell mass studies were performed. Control cells were exposed to the respective solvent (water).
4
Caco-2 Monolayer Permeability Assay
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Caco-2 cells were purchased from the American Type Culture Collection (ATCC) and grown in MEM supplemented with 200 ml/L of FBS, 10 IU/ml of penicillin, and 10 mg/l streptomyocin. The cells were kept at 37°C in a 5% CO2 environment. The cells were subcultured by partial digestion with 0.25% trypsin and EDTA. Passages 25–60 were used for experiements. For monolayer experiments, Caco-2 cells were grown on Corning BioCoaT HTS 1.0 μm filter support transwell plaes in Basal Seeding Medium (BSM) for 2 days and Entero-STIM Enterocyte Differentiation Medium (EDM) for 1 day. Both the BSM and EDM were supplmented with MITO+ Serum Extender according to the manufacturer’s protocol. The transepithelial electrical resistance (TEER) was measured with a Millicell Voltohmmeter to confirm the existance of a viable monolayer. Only Caco-2 monolayers with TEER values above 300 Ωcm2 were used for experiments.
5
Antimicrobial and Anticancer Evaluation
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Analytical reagent grade chemicals, including ferric chloride anhydrous, sulphuric acid, nitric acid, and hydrochloric acid, were procured from Loba Chemie Private Limited, Mumbai, India. Gum acacia powder, Meuller Hinton’s Agar, potato dextrose agar, Dulbecco’s Modified Eagles Medium, L-glutamine, and streptomycin (sulfate salt) were purchased from Sigma Aldrich, St. Louis, MO, USA. Ciprofloxacin, nutrient agar, nutrient broth, and streptomycin were procured from Hi-Media Private Limited, Mumbai, India. Gram-positive and Gram-negative food pathogenic microbial strains, i.e., Staphylococcus aureus (MTCC 3160) and Escherichia coli (MTCC 443), were obtained from the Microbial Type Culture Collection (MTCC), Institute of Microbial Technology, Chandigarh, India. Human colon adenocarcinoma cells (Caco-2 cells) were collected from National Centre for Cell Science, Pune, Maharashtra, India. Plastic dishes, plates, and transwell were obtained from Corning (Corning, NY, USA). Cellular grade, triple distilled water and aqua-regia washed glassware (class “A” certified) were used during research work.
6
Cytokine and Tight Junction Expression
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RAW264.7 and Caco-2 cells were bought from ATCC. Cultures were performed respectively in RPMI1640 medium or DMEM in a 6 well plate (NEST Biotechnology, Wuxi, China), and both media were supplemented with 10–20% heat-inactivated FBS, 50 U/ml penicillin and 50 U/ml streptomycin (Invitrogen, USA) in a 37 °C incubator containing 5% CO2. RAW264.7 cells were trypsinized and resuspended at 1 × 106 cells/ml for the subsequent determination of cytokine gene expression. For the lipopolysaccharide (LPS) experiments, cells were treated with 1.0 µg/ml LPS (Sigma, USA) from Escherichia coli 0:55:B55 diluted in ddH2O for 12 h. In the NZ9000 and NZ9000SHD-5 groups, RAW264.7 cells were treated with LPS and supernatants from NZ9000 or NZ9000SHD-5 cells for 12 h. Caco-2 cells were plated at a density of 1 × 106 cells/ml on collagen-coated permeable polycarbonate membrane Transwell supports with 0.3 µm pores (Corning, USA) and were grown as monolayers for subsequent measurement of TJ gene and protein expression.
7
Caco-2 Permeability Assay for Bile Acids
8
Caco-2 Cell Culture for Electrophysiology
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Caco-2 cells [American Type Culture Collection (ATCC) no. HTB -37] were cultured in a 25-cm 2 T-flask (Corning, NY) containing Dulbecco's modified Eagle's medium (DMEM; Sigma, St. Louis, MO) supplemented with 15% fetal bovine serum (GIBCO, Grand Island, NY), 1% nonessential amino acid (NEAA; Sigma), 1% L-glu- tamine (GIBCO), and 100 U/ml penicillin-streptomycin (GIBCO). Cells were subcultured as recommended by ATCC and maintained at 37°C under a humidified 5% CO 2 in an incubator. For the electrophysiological analysis, 5 ϫ 10 5 cells/well of Caco-2 cells (passage nos. [27] (link)[28] (link)[29] (link)[30] (link)[31] (link)[32] (link)[33] (link)[34] [35] (link) were grown on polyester Snapwells (12-mm diameter and 0.4-m pore size; Corning) and culture medium was changed daily for 14 days. Since the PTH responses of cells with passage nos. Ͼ40 may be slightly different from those of earlier passage numbers, we used cells with passage nos. Յ35 in the present study.
9
Caco-2 Cell Culture and Transport
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Caco-2 cells were obtained from the American Type Culture Collection (Manassas, VA, USA). Cells were cultured on tissue culture flasks in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum (Corning, NY, USA) and 1% penicillin streptomycin (Corning, NY, USA). These cells were maintained in an atmosphere of 5% CO2/95% air at 37 °C. The culture medium was refreshed every 3 to 5 days after detaching with trypsin-EDTA. For transport experiments, Caco-2 cells with passage number 49 were seeded into 12-well Transwell plates (1.12 cm2 in surface, 0.4 μm in pore size, 12 mm in diameter, Corning Costar Corporation, Cambridge, MA, USA) at a density of 3.5 × 105 cells/insert and cultured for 3 weeks. The integrity of cell monolayer was evaluated prior to transport experiments by measuring transepithelial electrical resistance (TEER). TEER values greater than 200 Ωcm2 for Caco-2 cell monolayers were used in the transport experiment.
10
Caco-2 Cells Barrier Integrity Assay
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Human intestinal epithelial Caco-2 cells (ATCC, Virginia, USA) were seeded in 12-well transwell plates containing an apical chamber made of polyester membrane filters with 0.4 µm pore size (Corning) at a density of 3×105/well. The culture medium from both the apical and basolateral compartments was changed every 2 days. The cells are allowed to fully differentiate for 21 days, and fully differentiated cells with no leakage tested were challenged with FCM and metabolites for the next 8 hours with continuous measuring of TEER values, using an EVOM2 Epithelial Voltmeter (WPI, Florida, USA). The blank inert resistance value (the insert with only culture media) was subtracted from the measured resistance value of each sample, and the final resistance in ohm × cm2 was calculated by multiplying the sample resistance by the area of the membrane.
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