Caco-2 cells and HEK293 cells were obtained from American Type Culture Collection (ATCC). Caco-2 cells were grown routinely in Eagle’s minimum essential medium (EMEM) (ATCC) supplemented with 10% fetal bovine serum (FBS) while HEK293 cells were grown routinely in Dulbecco’s modified Eagle’s medium (DMEM) (ATCC) supplemented with 10% FBS. Caco-2 and HEK293 cells were maintained in 5% CO2–95% air at 37C. Cell lines were routinely tested for mycoplasma using a commercially available kit (Lonza) according the manufacturer’s instructions.
Caco 2 cell
Manufactured by American Type Culture Collection
Sourced in United States, United Kingdom, Germany, France
Caco-2 cells are a continuous cell line derived from human colorectal adenocarcinoma. They are commonly used as an in vitro model for studying intestinal absorption and permeability of drugs and other compounds.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (63 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (34 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (24 mentions)
Penicillin, by Thermo Fisher Scientific (15 mentions)
Streptomycin, by Thermo Fisher Scientific (14 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (14 mentions)
Fetal bovine serum (fbs), by Merck Group (10 mentions)
L glutamine, by Thermo Fisher Scientific (10 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (9 mentions)
Caco 2 cells, by Thermo Fisher Scientific (8 mentions)
Hek293 cell, by American Type Culture Collection (7 mentions)
Transwell plate, by Corning (7 mentions)
Hek293t cell, by American Type Culture Collection (6 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (6 mentions)
Vero e6 cell, by American Type Culture Collection (6 mentions)
Penicillin and streptomycin, by Thermo Fisher Scientific (6 mentions)
Thp 1 cell, by American Type Culture Collection (6 mentions)
Penicillin streptomycin, by Merck Group (6 mentions)
Microaerophilic system, by Thermo Fisher Scientific (6 mentions)
Hepg2 cell, by American Type Culture Collection (5 mentions)
349 protocols using caco 2 cell
1
Culturing Caco-2 and HEK293 Cells
2
Rabbit Antibody Inhibition of ETEC Adherence
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Rabbit serum antibody in vitro activity against adherence from E. coli or ETEC bacteria expressing the seven adhesins targeted by MecVax or CFA/I/II/IV MEFA protein was measured in adherence inhibition assays with Caco-2 cells (ATCC no. HT-37). As described previously (17 (link), 24 (link), 44 (link), 46 (link)), E. coli recombinant strains expressing CS1 or CS2 or ETEC strains producing CFA/I, CS3, CS4/CS6, CS5/CS6, or CS6 adhesin (1.25 × 104 bacteria), after incubation with 15 μL heat-inactivated rabbit serum sample from the group injected with MecVax, CFA/I/II/IV MEFA protein, or PBS for 30 min at room temperature (50 rpm), were added to 95% to 100% confluent Caco-2 cells (ATCC HTB-37) cultured in 48-well plate wells, at a multiplicity of infection (MOI) of 10 bacteria per cell. After incubation in a 37°C CO2 incubator for 1 h, cells were washed with PBS (to remove nonadherent bacteria), dislodged, and harvested. Collected materials were serially diluted, plated on MacConkey agar plates, and cultured 37°C overnight. E. coli or ETEC bacteria (CFU) were counted and recorded. Bacterial adherence was presented in percentage, with bacteria (CFU) adherent to Caco-2 cells treated with the control rabbit serum as 100%; subtraction of bacterial adherence percentage from 100% was referred to as adherence inhibition or reduction.
3
Rabbit Antibody Inhibition of ETEC Adherence
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Rabbit serum antibody in vitro activity against adherence from E. coli or ETEC bacteria expressing the seven adhesins targeted by MecVax or CFA/I/II/IV MEFA protein was measured in adherence inhibition assays with Caco-2 cells (ATCC no. HT-37). As described previously (17 (link), 24 (link), 44 (link), 46 (link)), E. coli recombinant strains expressing CS1 or CS2 or ETEC strains producing CFA/I, CS3, CS4/CS6, CS5/CS6, or CS6 adhesin (1.25 × 104 bacteria), after incubation with 15 μL heat-inactivated rabbit serum sample from the group injected with MecVax, CFA/I/II/IV MEFA protein, or PBS for 30 min at room temperature (50 rpm), were added to 95% to 100% confluent Caco-2 cells (ATCC HTB-37) cultured in 48-well plate wells, at a multiplicity of infection (MOI) of 10 bacteria per cell. After incubation in a 37°C CO2 incubator for 1 h, cells were washed with PBS (to remove nonadherent bacteria), dislodged, and harvested. Collected materials were serially diluted, plated on MacConkey agar plates, and cultured 37°C overnight. E. coli or ETEC bacteria (CFU) were counted and recorded. Bacterial adherence was presented in percentage, with bacteria (CFU) adherent to Caco-2 cells treated with the control rabbit serum as 100%; subtraction of bacterial adherence percentage from 100% was referred to as adherence inhibition or reduction.
4
Caco-2 Cell Differentiation Protocol
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Caco-2 cells (ATCC, MD, USA) were grown on minimal essential medium (MEM) (Gibco, MA, USA) containing 10% fetal bovine serum, 1% GlutaMAX (Gibco, MA, USA), 1% MEM nonessential amino acids (Gibco, MA, USA), and 1% sodium pyruvate at 37°C and 5% CO2. After reaching confluence, the cells were seeded into 6- or 96-well plates at a density of 1 × 105 cells/well for individual experiments and then cultured for 21 days (on average), with the medium changed every other day until the cells had differentiated.
5
Zika Virus Infection in Caco-2 Cells
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Caco-2 cells (ATCC) were maintained in minimal essential medium (MEM; Invitrogen) supplemented with 2% or 10% fetal bovine serum (Invitrogen). Zika virus PR (Puerto Rico; GenBank KX087101.3; passage 3 and 4) was used for all the experiments and titers were determined with plaque assay performed on Vero-E6 cells.
6
Caco-2 Cell Viability Assay
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The device was precoated with 860 μg/mL rat tail type I collagen (BD Biosciences, CA, USA) diluted in 0.02 N acetic acid. Caco-2 cells (ATCC, VA, USA) were perfused in a concentration of 14 million cells/mL to the device at a flow rate of 25 µL/min. Cells were incubated in the device with daily media changes for 7 days in a 5% CO2-humidified incubator at 37°C. In day 7, viability of cells was quantified using LIVE/DEAD viability assay (Invitrogen Life Sciences, CA, USA), in which the cytoplasm of live cells accumulates green-fluorescent calcein because of esterase activity, while the nuclei of dead cells are labeled red by ethidium homodimer owing to a loss of nuclear membrane integrity. Cells were quantified using ImageJ.
7
Cell Culture Characterization and Maintenance
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Tissue culture medium and dialyzed fetal bovine serum (FBS) were from Invitrogen (Carlsbad, CA), and biochemicals were from Sigma-Aldrich (St. Louis, MO). The antibodies recognizing CELF1, HuR, Myc, GAPDH, and β-actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and BD Biosciences. The secondary antibody conjugated to horseradish peroxidase was purchased from Sigma-Aldrich. The IEC-6 cell line (derived from normal rat intestinal crypt cells) was purchased from the American Type Culture Collection (ATCC) at passage 13 and was maintained in T-150 flasks in DMEM supplemented with 5% heat-inactivated FBS. Passages 15–20 were used in experiments, and there were no significant changes of biological function and characterization of IEC-6 cells at passages 15–20 (Li et al., 2001 (link); Zhang et al., 2004 (link); Wang et al., 2010 (link)). Caco-2 cells (a human colon carcinoma cell line) were also purchased from ATCC and cultured similarly to the IEC-6 cells.
8
Caco-2 Monolayer Permeability Assay
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Caco-2 cells (ATCC, Manassas, VA) were grown on 24-well (pore size: 0.4 μm) polycarbonate filters. The monolayers were pre-incubated with pre-warmed HBSS (Hank’s balanced salt solution) containing 2.5% HEPES buffer (pH 7.4) for 0.5 h at 37ºC. After pre-incubation, the buffer was removed and pyronaridine was added to reach a final concentration of 10 μM. 2% bovine serum albumin (BSA) was added to the receiver buffer for the study. The total volume was 400 μL for the apical (A) side and 1200 μL for the basolateral (B) side. For apical to basolateral transport study (A-B), 100 μL each was collected from both sides for sample analysis at the start of the assay and then 200 μL was collected from the apical side at 90 minutes (end of the study). The same timepoints and amounts were used for the basolateral to apical transport study (B-A).
The apparent permeability coefficient (Papp) was calculated from the following equation.
Where:
v = Volume of the receiver cell
A = Exposed surface area (0.64 cm2)
C = Initial donor concentration
∂C/∂t = Change in receiver concentration over time.
The apparent permeability coefficient (Papp) was calculated from the following equation.
Where:
v = Volume of the receiver cell
A = Exposed surface area (0.64 cm2)
C = Initial donor concentration
∂C/∂t = Change in receiver concentration over time.
9
Caco-2 Cell-based Permeability Assay
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Caco-2 cells were procured from ATCC, USA. Transwell 24-well inserts were procured from Corning (USA), Dulbecco’s modified eagles medium (DMEM) was from Gibco, 96 well plate, hydrophilic solvinert plates were purchased from Millipore, USA. Digoxin, quinidine, quercetin, silymarin and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (USA) and all other chemicals were of HPLC grade.
10
Caco-2 Cell Culture and Infection Protocol
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