The 3-D model system comprised of the human intestinal epithelial cell line HCT-8 cells (CCL-244, ATCC, Manassas, VA) and primary human lymphocytes/monocytes, endothelial cells (HUVEC cells, CRL-1459, ATCC), and fibroblasts (CCD-18Co cells, CRL-1459, ATCC) was cultured under microgravity conditions. Cell cultivation and the set-up of the 3-D model were performed as previously described (8 (link)–10 (link), 17 (link)). Briefly, fibroblasts and endothelial cells were embedded in an enriched collagen-I matrix (Invitrogen, Carlsbad, CA) and added together with epithelial cells to the rotating wall vessels (RWV) (Synthecon, Houston, TX) that provided microgravity conditions to the cultures. Peripheral blood mononuclear cells (PBMC) enriched in lymphocytes/monocytes isolated from healthy adult volunteers (2 × 107/vessel) were added to the 3-D model culture at days 4 and 9 (± 1 days) (8 (link)–10 (link), 17 (link)). PBMC were isolated by Ficoll density gradient centrifugation, and added to the cultures without stimulation. Thus, these PBMC have the same frequencies of cell subsets as those observed in circulation in healthy adults, e.g., 70–90% lymphocytes, 10–20% monocytes and 1–2% dendritic cells (18 ). The experiments were performed with 15–18 days-old 3-D models after the cells within the 3-D model were fully differentiated.
Crl 1459
Manufactured by American Type Culture Collection
Sourced in United States, Holy See (Vatican City State)
CRL-1459 is a cell line derived from human lung carcinoma. It is a widely used research tool for studying various aspects of cancer biology and drug development.
Automatically generated - may contain errors
Lab products found in correlation
Ccd 18co, by American Type Culture Collection (4 mentions)
Hct116, by American Type Culture Collection (3 mentions)
Ccl 247, by American Type Culture Collection (2 mentions)
Hct116, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Dharmafect 2, by Horizon Discovery (1 mentions)
Wnt c59, by Selleck Chemicals (1 mentions)
Recombinant human tgf β1, by R&D Systems (1 mentions)
Sw620, by Thermo Fisher Scientific (1 mentions)
D5030, by Merck Group (1 mentions)
Ccl 227, by American Type Culture Collection (1 mentions)
Hct15, by Thermo Fisher Scientific (1 mentions)
Ccl 228, by American Type Culture Collection (1 mentions)
Ccl 229, by American Type Culture Collection (1 mentions)
Ccl 225, by American Type Culture Collection (1 mentions)
Ccd 18co, by Merck Group (1 mentions)
Sw480, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
12 protocols using crl 1459
1
Microgravity-Cultured 3D Intestinal Model
2
Modulating Wnt and TGFβ Signaling
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CCD-18Co (normal human intestinal fibroblasts, ATCC® CRL-1459™) were cultured according to guidelines. For all cell treatments, cells were serum starved overnight prior to 48 h treatment with 10 µM ICG-001 dissolved in DMSO, or 100 nM porcupine inhibitor Wnt-C59 (Selleckchem S7037), or 10 μM FZD8 inhibitor 3235-0367 (C1, ChemDiv), alone or in combination with 10 ng/mL-recombinant human TGFβ1 (R&D Systems). For FZD8 siRNA-mediated gene knockdowns in 96-well plates, cells were transfected with either a siRNA-targeted against FZD8 (25 nM, SC-39992) or a nontargeted control (SC-37007) using DharmaFECT 2 reagent (T-2002-02). Transfections were performed according to the Dharmacon™ DharmaFECT™ 1–4 transfection protocol, using 0.1 μL of DharmaFECT 2 per reaction. For protein analysis, siRNA protocols were scaled-up proportionally.
3
Colon Cancer Cell Lines Protocol
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Human CRC cells (HCT116, HCT15, SW480, SW620, and LOVO) and human normal colon cells (CCD‐18Co) were purchased from American Type Culture Collection (CCL‐247, CCL‐225, CCL‐228, CCL‐227, CCL‐229, and CRL‐1459, ATCC). HCT15, SW480, and SW620 cells were maintained in Roswell Park Memorial Institute (RPMI)‐1640 media (A4192301, ThermoFisher). HCT116, LOVO, and CCD‐18Co cells were cultured in Dulbecco's modified Eagle's media (DMEM; D5030, Sigma‐Aldrich). All the media were made to be complete media by the addition of 10% fetal bovine serum (FBS, F2442, Sigma‐Aldrich) and 1% penicillin–streptomycin (P4333, Sigma‐Aldrich). Cell culture was performed in humidified atmosphere containing 5% CO2 at 37°C.
4
Culturing human colonic epithelial and fibroblast cells
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The human colonic epithelial cell lines 1CT were previously described (25 (link)). 1CT cells were cultured in complete medium, composed as follows: high‐glucose Dulbecco’s Modified Eagle (DMEM) medium/medium 199 (4530, Sigma‐Aldrich Merck, Darmstadt, Germany, at ratio 4:1), supplemented with 2% fetal bovine serum (FBS; 10270-106, Gibco ThermoFisher Scientific, Waltham, MA, USA), epidermal growth factor (EGF; 20 ng/ml, E9644, Sigma‐Aldrich Merck), hydrocortisone (1 mg/ml, H0396, Sigma‐Aldrich Merck), insulin (10 mg/ml, I9278, Sigma‐Aldrich), transferrin (2 mg/ml, T0665, Sigma‐Aldrich Merck), sodium selenite (5 nM, S5261, Sigma‐Aldrich Merck), and geneticin (G418 sulfate) sulfate (50 μg/ml, G1397, Sigma‐Aldrich Merck).
Human colonic fibroblasts (CRL-1459, ATCC, Manassas, VA, USA) were maintained in Eagle’s Minimum Essential Medium (EMEM; 30-2003, ATCC, Manassas, VA, USA) supplemented with 10% of FBS (10270-106, Gibco, ThermoFisher Scientific) and 10 μg/ml of ciprofloxacin (17850, Sigma‐Aldrich Merck).
Human colonic fibroblasts (CRL-1459, ATCC, Manassas, VA, USA) were maintained in Eagle’s Minimum Essential Medium (EMEM; 30-2003, ATCC, Manassas, VA, USA) supplemented with 10% of FBS (10270-106, Gibco, ThermoFisher Scientific) and 10 μg/ml of ciprofloxacin (17850, Sigma‐Aldrich Merck).
5
Cultivation of Human Colon Fibroblasts
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The primary/early-passage normal human female colon fibroblast CCD-18Co cell line, with an approximate population doubling time of 42 hours, was purchased from the American Type Culture Collection (CRL-1459, ATCC, Maryland, USA). CCD-18Co cells were cultured in minimal essential medium (MEM, Gibco, Carlsbad, CA, USA) supplemented with 15% fetal bovine serum (FBS, Gibco), 1% nonessential amino acids (100 nmol/L, Thermo Fisher Scientific, Waltham, USA), and 100 U/mL penicillin/streptomycin (Invitrogen, CA, USA) and maintained at 37°C in humidified air with 5% CO 2 . Cells were expanded six times and then stored in liquid nitrogen or used in experiments. The cell line was tested and authenticated by short tandem repeat (STR)
6
Colorectal Cancer Cell Lines Cultivation
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Two colon cancer cell lines were used for the study: HCT-116 (ATCC® CCL-247™, Manassas, VA, USA) and DLD-1 (ATCC® CCL-221™, Manassas, VA, USA) and one normal fibroblast cell line: CCD-18Co (ATCC® CRL-1459™, Manassas, VA, USA). The cancer cell lines are originating from colorectal patients, although the transformed cells are from the colon biopsy. HCT-116 cell line was cultured in Mc Coy’s media (Merck KGaA, Darmstadt, Germany) supplemented with 10% fetal bovine serum (FBS), DLD-1 cell line was cultured in RPMI media (Merck KGaA, Darmstadt, Germany) supplemented with 10% FBS, while CCD-18Co cell line was cultured in MEM media (Merck KGaA, Darmstadt, Germany) supplemented with 10% FBS according to the provider protocol. All cell lines were passaged 2–3 times a week and were incubated at 37 °C with 5% CO2. The 3D spheroids of DLD-1 were obtained through the hanging drops technique as previously exemplified [17 (link)].
7
Culturing CCD-18Co and Caco-2 Cell Lines
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The research subjects were
CCD-18Co (ATCC CRL-1459) and CaCo-2 (ATCC HTB-37) cell lines purchased
from ATCC: The Global Bioresource Center. The CCD-18Co cell line was
cultured in accordance with the manufacturer’s recommendations
using ATCC-formulated Eagle’s minimum essential medium withl -glutamine (catalog No. 30-2003). To make the complete growth
medium, fetal bovine serum was added to a final concentration of 10%.
The complete culture medium was renewed every 2–3 days. The
cells CCD-18Co were obtained from a patient, and their characteristics
and morphology are normal myofibroblasts in the colon. The CaCo-2
cell line was also cultured according to the ATCC protocols. The CaCo-2
cell line was obtained from a patient—a 72-year-old Caucasian
male diagnosed with colon adenocarcinoma. To make the medium complete,
we based it on Eagle’s minimum essential medium withl -glutamine (catalog No. 30-2003), with the addition of a fetal bovine
serum to a final concentration of 20%. The medium was renewed once
or twice a week.
The biological safety of both CCD-18Co and
CaCo-2 cell lines has been classified by the American Biosafety Association
(ABSA) as level 1 (BSL-1).
CCD-18Co (ATCC CRL-1459) and CaCo-2 (ATCC HTB-37) cell lines purchased
from ATCC: The Global Bioresource Center. The CCD-18Co cell line was
cultured in accordance with the manufacturer’s recommendations
using ATCC-formulated Eagle’s minimum essential medium with
medium, fetal bovine serum was added to a final concentration of 10%.
The complete culture medium was renewed every 2–3 days. The
cells CCD-18Co were obtained from a patient, and their characteristics
and morphology are normal myofibroblasts in the colon. The CaCo-2
cell line was also cultured according to the ATCC protocols. The CaCo-2
cell line was obtained from a patient—a 72-year-old Caucasian
male diagnosed with colon adenocarcinoma. To make the medium complete,
we based it on Eagle’s minimum essential medium with
serum to a final concentration of 20%. The medium was renewed once
or twice a week.
The biological safety of both CCD-18Co and
CaCo-2 cell lines has been classified by the American Biosafety Association
(ABSA) as level 1 (BSL-1).
8
Colon Cancer Cell Lines and Knockdown
9
Isolation and Culture of Colon Fibroblasts
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Fibroblasts were isolated from primary tissues using previously described methodologies [3 (link)]. Briefly, after mincing, heated collagenase digestion on a shaking water bath was used and the filtrate was cultured on tissue culture plates in DMEM 10% FBS (Atlanta Biologicals) with 1% Pen/Strep (Gibco). Human colon fibroblasts cell lines CRL1541, CRL1459, and CRL7213 were obtained from the American Type Culture Collection (ATCC). Primary colon fibroblasts were isolated from human colon tissue was obtained with informed consent from patients undergoing surgery without preoperative treatment for sporadic colon cancer or colectomy for chronic ulcerative colitis. Primary fibroblasts were cultured from non-dysplastic sections of colon (normal), adenocarcinoma or colitic sections as previously described [3 (link)].
10
Culturing of Colon Myofibroblast Cell Line
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