The integrity of monolayer enterocytes in different conditions was determined by TEER using Caco-2 cells (HTB-37) (ATCC) at 5 × 104 cells per on the upper compartment of 24-well Boyden chamber trans-well plate in DMEM-high glucose supplemented with 20% fetal bovine serum (FBS), 1% HEPES, 1% sodium pyruvate and 1.3% Penicillin/Streptomycin for 15 days to establish the confluent monolayer. Then, LPS from E. coli O26:B6 (Sigma-Aldrich) at 100 ng/mL with or without BG (Pachyman) (Megazyme) at 100 µg/mL were incubated for 4 h before TEER determination using an epithelial volt-ohm meter (EVOM-2, World precision instruments, Florida, USA) by placing the electrodes in the supernatant at the basolateral and apical chamber. The TEER value in media culture without cells was used as a blank and was subtracted from all measurements. The unit of TEER was ohm (Ω) × cm2.
Caco 2 cells htb 37
Manufactured by American Type Culture Collection
Sourced in United States
Caco-2 cells (HTB-37) are a human epithelial colorectal adenocarcinoma cell line derived from the colon of a 72-year-old Caucasian male. These cells exhibit several morphological and functional characteristics of the intestinal columnar epithelium and are widely used as an in vitro model for the study of intestinal absorption and transport mechanisms.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (7 mentions)
Trypsin edta, by Thermo Fisher Scientific (6 mentions)
Normocin, by InvivoGen (3 mentions)
Transwell polycarbonate insert, by Corning (2 mentions)
Penicillin streptomycin solution, by Merck Group (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Penicillin streptomycin, by Merck Group (2 mentions)
Lps from e coli o26 b6, by Merck Group (1 mentions)
Evom2, by World Precision Instruments (1 mentions)
Hbss buffer, by Thermo Fisher Scientific (1 mentions)
Hepes, by Merck Group (1 mentions)
Penicillin streptomycin, by Lonza (1 mentions)
L glutamine, by Lonza (1 mentions)
Dmem medium, by Lonza (1 mentions)
Fluorescein isothiocyanate conjugated dextran 4 kda fd 4, by Merck Group (1 mentions)
Tnf α, by Merck Group (1 mentions)
Trypsin, by Solarbio (1 mentions)
Alcalase, by Solarbio (1 mentions)
Papain, by Solarbio (1 mentions)
17 protocols using caco 2 cells htb 37
1
Caco-2 Monolayer Integrity Assay
2
GLP-2 receptor cell assay protocol
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Resin and natural amino acids were purchased at Novabiochem (Germany). c8, c12 and c16 carboxylic acids, Fmoc-beta-Alanine and native GLP-2 were provided by Novo Nordisk A/S. Palmitoyloleoylglycerophosphocholine (POPC) was purchased from Avanti Polar Lipids (USA). HEPES, Ovalbumin (OVA, from chicken egg white) and other standard chemicals were purchased from Sigma-Aldrich (Denmark). DMEM medium, l-glutamine and penicillin/streptomycin was purchased from Lonza (Switzerland). HBSS buffer, fetal bovine serum (FBS), nonessential amino acid and other standard cell culture products were purchased from Gibco (Denmark). Radioactively labeled [ ]mannitol, scintillation fluid (Microscint-40), luciferase substrate (SteadyLite) and 96-well plates for luciferase assay (CulturPlate, black) were purchased from PerkinElmer (USA). 12 well Transwell plates for Caco-2 cell monolayers (polycarbonate, 12 mm, pore size 0.4 ) were purchased from Corning Costar Corp. (USA). GLP-2R BHK cells were provided by Novo Nordisk (the cloning was previously described by Thulesen et al. [26] (link) and Sams et al. [27] (link)) and Caco-2 cells (HTB-37) were purchased from ATCC.
3
Collagen Extraction and Cell Culture Protocol
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Alaska pollock skin-derived collagen was obtained from Dongyi Tech Co., Ltd (Qingdao, China). Caco-2 cells (HTB-37) were purchased from ATCC (Rockville, MD, USA). All cell culture reagents and supplies were purchased from Thermo Fisher Scientific Co., Ltd. (Shanghai, China). Alcalase, flavourzyme, papain, and trypsin were purchased from Beijing Solarbio Science and Technology Co., Ltd., (Beijing, China). TNF-α, fluorescein isothiocyanate-conjugated dextran 4 kDa (FD-4), and all other reagents used in this study without attribution were obtained from Sigma-Aldrich Chemical Co. (Shanghai, China).
4
Caco-2 Cell Culture and Osmolarity Regulation
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Human epithelial colorectal adenocarcinoma Caco‐2 cells (HTB‐37) were purchased (ATCC, Manassas, VA) and were cultured in Dulbecco's Modified Eagle Medium (DMEM) with a high d ‐glucose content (4.5 g/L), containing l ‐glutamine, and supplemented with 10% heat‐inactivated fetal bovine serum (Life Technologies, Dublin, Ireland). Once cells reached 90% confluency, they were subcultured in a 1:6 dilution every 5 days. Media were removed and replaced with fresh media every 2–3 days. Cells were incubated at 37°C with 5% CO2. For regulatory experiments, cells which had reached 90% confluency were exposed to media containing additional NaCl, mannitol, or butyrate for 24 h prior to RNA extraction. Osmolalities of different experimental media were confirmed using an Osmomat 030 osmometer (Gonotec, Germany) and measurements (mean ± standard error) were as follows: “250 mOsm” = 246 ± 22 mOsm (N = 4); Control “350 mOsm” = 354 ± 13 mOsm (N = 5); “500 mOsm” = 502 ± 9 mOsm (N = 3); “600 mOsm” with NaCl = 595 ± 30 mOsm (N = 5); and “600 mOsm” with mannitol = 634 ± 15 mOsm (N = 4).
5
Epithelial Cell Priming and Infection
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Where indicated, cells were primed with 100ng/ml or 400ng/ml of Pam3CSK4 (Invivogen) for 3 hours prior to infection. To induce SPI-1 expression, overnight cultures of Salmonella were diluted into LB broth containing 300 mM NaCl and grown for 3 hours standing at 37°C (28) . Overnight cultures of Listeria were diluted and grown shaking for Cell culture of intestinal epithelial cell lines All cell lines were obtained from American Type Culture Collection (ATCC).
Caco-2 cells (HTB-37; ATCC) were maintained in DMEM supplemented with 10% (vol/vol) heat-inactivated FBS, 100 IU/mL penicillin, and 100 μg/mL streptomycin. T84 cells (CCL-248; ATCC) were maintained in DMEM F-12 supplemented with 5% (vol/vol) heat-inactivated FBS, 100 IU/mL penicillin, and 100 μg/mL streptomycin.
One day prior to infection or treatment, cells were dissociated with 0.25% Trypsin-EDTA (Gibco) diluted 1:1 with 1X PBS. Cells were incubated with trypsin at 37°C for 15 minutes, after which the trypsin was neutralized with serum-containing media. Cells were replated in media without antibiotics in a 24-well plate at a concentration of 3 × 10 5 cells/well. Where indicated, cells were primed with 100ng/mL or 400ng/ml Pam3CSK4 (Invivogen) or 500ng/ml LPS (Sigma-Aldrich) for 3h or 16h prior to anthrax toxin treatment or bacterial infections.
Caco-2 cells (HTB-37; ATCC) were maintained in DMEM supplemented with 10% (vol/vol) heat-inactivated FBS, 100 IU/mL penicillin, and 100 μg/mL streptomycin. T84 cells (CCL-248; ATCC) were maintained in DMEM F-12 supplemented with 5% (vol/vol) heat-inactivated FBS, 100 IU/mL penicillin, and 100 μg/mL streptomycin.
One day prior to infection or treatment, cells were dissociated with 0.25% Trypsin-EDTA (Gibco) diluted 1:1 with 1X PBS. Cells were incubated with trypsin at 37°C for 15 minutes, after which the trypsin was neutralized with serum-containing media. Cells were replated in media without antibiotics in a 24-well plate at a concentration of 3 × 10 5 cells/well. Where indicated, cells were primed with 100ng/mL or 400ng/ml Pam3CSK4 (Invivogen) or 500ng/ml LPS (Sigma-Aldrich) for 3h or 16h prior to anthrax toxin treatment or bacterial infections.
6
Caco-2 Cell Viability Assay with LPS and Glycyrrhizic Acid
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Human colorectal adenocarcinoma epithelial cells, Caco-2 cells (HTB-37), were purchased from American Type Culture Collection (ATCC, Manassas, VA), and cultured in ATCC-formulated Eagle’s Minimum Essential Media (30-2003, ATCC, Manassas, VA) supplemented with 20% foetal bovine serum (FBS, F2442, Sigma-Aldrich, St. Louis, MO) at 37 °C with 5% CO2. GP (HY-N6881, C41H70O12, purity: ≥90.0%, CAS no. 80325-22-0, MedChemExpress, Monmouth Junction, NJ) were solubilized in dimethyl sulphoxide (D2650, DMSO, Sigma-Aldrich, St. Louis, MO), and diluted into the gradient concentrations of 0, 50, 100, 150 or 200 μM for the use in drug treatment. Notably, DMSO was set at the 0.1% final concentration in in vitro experiments. Also, gradient GP were applied in the measurement of cell viability. Then, Caco-2 cells were assigned into four groups (control group, LPS group, LPS + GP150 group and LPS + GP200 group). Caco-2 cells in the LPS + GP150 group or the LPS + GP200 group were treated with 150 μM or 200 μM of GP at 37 °C for 24 h with 5% CO2. After being washed with phosphate-buffered saline (PBS, P5493, Sigma-Aldrich, St. Louis, MO), cells were exposed to 10 μg/mL LPS (HY-D1056, LPS, MedChemExpress, Monmouth Junction, NJ) at 37 °C for 24 h with 5% CO2 (He et al. 2020 (link)). Cells in the LPS group only underwent 10 μg/mL LPS exposure for 24 h, and cells in the Control group remained untreated.
7
Caco-2 Cell Culture and Transwell Assay
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Caco-2 cells (HTB-37) were obtained from the American Type Culture Collection (Rockville, MD, USA) at passage 20. Stock cultures were maintained in Dulbecco’s modified Eagle’s medium supplemented with fetal bovine serum (FBS; 16%) and non-essential amino acids (0.9%). The medium contained an agent for the prevention of bacterial, fungal, or mycoplasma infection; Normocin (Invivogen, San Diego, CA, USA). The cell cultures were incubated at 37 °C in 95% humidified air and 5% CO2. The medium was changed every second or third day and the cells were passaged at approximately 80% confluence. At passage 29–39, the cells were seeded in 12-well plates with Transwell® polycarbonate inserts (Corning, San Fransisco, MA, USA (0.4 μm) at 60,000 cells/insert or without inserts at 200,000 cells/well. All experiments were carried out 14 days after seeding. Aspirated medium samples were streaked on tryptone glucose extract agar plates to verify the absence of bacterial/fungal infections.
8
In vitro Gastrointestinal Digestion Assay
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All reagents used were of analytical grade. All solutions were prepared using ultra-high pure (UHP) 18 Ω water. All laboratory ware was soaked for 24 h in an acid bath containing 10% (v/v) nitric acid and then rinsed with UHP water. Concentrated hydrochloric acid (12 mol L−1), HNO3 69%, NaCl, NH4Cl, anhydrous Na2SO4, CaCl2.2H2O, NaHCO3, anhydrous D ( + )-glucose, sodium arsenate dibasic heptahydrate, cadmium acetate and lead acetate trihydrate were obtained from Fisher Scientific. d-glucuronic acid, Pancreatin (pig), pepsin (pig), Bovine serum albumin (BSA), KSCN, NaH2PO4, mucin (pig), D-glucosamine hydrochloride, lipase (pig), α-amylase (Bacillus species), urea and bile salts (bovine), modified eagle medium (DMEM) containing 4.5 g/L glucose, penicillin-streptomycin solution (10, 000 units penicillin and 10 mg streptomycin per mL), and Hank’s Balanced Salt Solution (HBSS) and glycine were obtained from Sigma–Aldrich (St. Louis, MO, USA). KH2PO4, MgCl2·6H2O, KCl, and uric acid were obtained from VWR. Caco-2 cells (HTB-37™) were purchased at passage 18 from the American Type Culture Collection (ATCC, Manassas, VA, USA). Fetal bovine serum and trypsin-EDTA were purchased from Invitrogen (Burlington, ON, Canada).
9
Maintaining Caco-2 Cell Line for Research
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The human colon carcinoma cell line Caco-2 cells (HTB-37) was obtained from the American Type Culture Collection (ATCC, LGC Promochem, Molsheim, France) at passage 21. Caco-2 cells were used between passage 25 and 30. The cells were maintained in 75 cm 2 culture flasks at 37°C under a humidified atmosphere (5% CO 2 : 95% O 2 ) in DMEM supplemented with 584 mg/L of L-glutamine, 1% (v/v) MEM, 100 U/ml of penicillin-streptomycin, 0.25 g/ml amphotericin B and 15% heat-inactivated fetal calf serum (FCS).
10
Caco-2 Cell Culture Protocol for Bioaccessibility Assays
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Caco-2 cells (HTB-37) were obtained from the American Type Culture Collection (Rockville, MD) at passage 19. Stock cultures were maintained in Dulbecco's modified essential medium (DMEM) supplemented with FBS (16%) and Normocin (InvivoGen, San Diego, USA). The cells were cultured at 37ºC/95% humidified air/5% CO2. The medium was changed every second or third day and the cells were passaged at 80-90% confluence. After passage, the cells were re-seeded at a density of 11,000 cells cm_2. For experiments, cells were seeded at passage 29 in 12-well plates at a density of 200,000 cells per well. All experiments were carried out 14 days post-seeding. The bioaccessibility assays were performed using the method of Meca et al. 22
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