Human insulin and FITC-insulin (Human) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Fluorescein isothiocyanate isomer I, 95% (FITC) was purchased from Alfa Aesar (Tewksbury, MA, USA). PD-10 Sephadex™ G-25 M columns were purchased from GE Healthcare (Buckinghamshire, UK). Dulbecco's modified Eagle's medium (DMEM), Hyclon Hank's 1X balanced salt solution (HBSS), penicillin-streptomycin solution and trypsin-EDTA solution were purchased from Fisher Scientific (Middletown, VA, USA). Transwell® inserts were purchased from VWR International (Allison Park, PA, USA). Human insulin ELISA kit was purchased from Crystal Chem (Elk Grove Village, IL, USA). MDCK cell line was purchased from ATCC® (Rockville, MD, USA).
Mdck cell line
Manufactured by American Type Culture Collection
Sourced in United States
The MDCK (Madin-Darby Canine Kidney) cell line is a well-established and commonly used in vitro model derived from the distal renal tubules of a normal adult female cocker spaniel. The MDCK cell line is characterized by its ability to form polarized monolayers and tight junctions, making it a valuable tool for studies related to epithelial cell biology, transport processes, and virus-host interactions.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Merck Group (3 mentions)
Penicillin streptomycin, by Merck Group (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Hank s balanced salt solution hbss, by Merck Group (2 mentions)
Mdck mdr1 cell lines, by Merck Group (2 mentions)
Mem non essential amino acid, by Merck Group (2 mentions)
Hepes, by Merck Group (2 mentions)
Acetonitrile, by Merck Group (2 mentions)
L glutamine, by Merck Group (2 mentions)
Trypsin edta, by Merck Group (2 mentions)
Dulbecco s modified eagle s medium dmem, by Merck Group (2 mentions)
Hplc grade solvent, by Merck Group (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Human insulin, by Merck Group (1 mentions)
Pd10 sephadex g25 m columns, by GE Healthcare (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin solution, by Thermo Fisher Scientific (1 mentions)
Trypsin edta solution, by Thermo Fisher Scientific (1 mentions)
Transwell inserts, by Avantor (1 mentions)
Human insulin elisa kit, by Crystal Chem (1 mentions)
16 protocols using mdck cell line
1
FITC-Insulin Permeability Assay
2
Influenza Virus Propagation and Detection Protocols
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The HEK 293T cell line (81 (link)) was obtained from J. C. de la Torre, the A549 human cell line (82 (link)) was obtained from J. A. Melero, and the Huh7 cell line (83 (link)) was obtained from P. Gastaminza. The MDCK cell line was purchased from the ATCC. Cell culture was carried out as previously described (84 (link)). Influenza virus strains A/Wisconsin/33 (H1N1) (WSN) and A/New Caledonia/20/99 (H1N1) and a recombinant virus containing the M, HA, and NA segments of WSN in the background of A/Victoria/3/75 (H3N2) (VIC) (85 (link)) were used in these experiments. Titrations were done in MDCK cells as previously described (86 (link)). VSV was provided by R. Alfonso and titrated in BHK21 cells. Plasmids pCMVPB1, pCMVPB2, pCMVPA, and pCMVNP, expressing the influenza virus polymerase subunits and the NP, have been previously described (85 (link)). Plasmid pHHclone23, a genomic plasmid expressing a deleted NS RNA segment (clone 23) (57 (link)) in negative polarity, was provided by R. Coloma. Antiserum specific for NP was generated by immunization of rabbits with recombinant NP (57 (link)). A polyclonal antibody specific for P-PERK (SC-32577) was purchased from Santa Cruz, and a monoclonal antibody specific for actin (ab8226) was purchased from Abcam. The secondary antibodies used for Western blotting were purchased from Sigma.
3
Permeability Assay of Drugs Using MDCK Cells
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The nine drugs tested (amitryptiline, atenolol, carbamazepine, fleroxacin, loperamide, norfloxacin, pefloxacin, propranolol and zolpidem) and HPLC grade solvents (acetonitrile, methanol and water) were purchased from Sigma-Aldrich (Barcelona, Spain). MDCK cell line was purchased from ATCC (USA) and MDCK-MDR1 cells were provided by Dr. Gottessman, MM (Nathional Institutes of Health, Bethesda). Pig brain homogenate was kindly supplied by a local slaughterhouse and fresh unfertilized chicken eggs were bought in a local supermarket. Table 2 shows the molecular properties of the nine drugs mentioned above.
Dulbecco’s modified Eagle’s medium (DMEM) with high content of glucose, L-glutamine, HEPES, MEM non-essential aminoacid, penicillin−streptomycin, trypsin-EDTA, Hank’s balanced salt solution (HBSS) and fetal bovine serum (FBS) for the cell culture of MDCK and MDCK-MDR1 cell lines were purchased from Sigma-Aldrich.
Dulbecco’s modified Eagle’s medium (DMEM) with high content of glucose, L-glutamine, HEPES, MEM non-essential aminoacid, penicillin−streptomycin, trypsin-EDTA, Hank’s balanced salt solution (HBSS) and fetal bovine serum (FBS) for the cell culture of MDCK and MDCK-MDR1 cell lines were purchased from Sigma-Aldrich.
4
In Vitro Drug Transport Model Validation
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The six drugs used for constructing the mathematical model (amitriptyline, caffeine, carbamazepine, fleroxacin, pefloxacin and zolpidem) and HPLC-grade solvents (acetonitrile, methanol and water) were purchased from Sigma-Aldrich (Barcelona, Spain). The MDCK cell line was purchased from ATCC (USA), MDCK-MDR1 cells were provided by Dr. Gottessman, and the MM (National Institutes of Health, Bethesda, MD, USA) and hCMEC/D3 cell lines were purchased from Cedarlane (Burlington, ON, Canada). Pig brain homogenate was kindly supplied by a local slaughterhouse.
Dulbecco’s modified eagle’s medium (DMEM) with a high content of glucose, L-glutamine, HEPES, MEM non-Essential aminoacid, penicillin−streptomycin, trypsin-EDTA, Hank’s balanced salt solution (HBSS) and fetal bovine serum (FBS) for the cell culture of MDCK and MDCK-MDR1 cell lines were purchased from Sigma-Aldrich (Barcelona, Spain).
The products needed for the culture of hCMEC/D3 cells were purchased from Sigma-Aldrich (Barcelona, Spain) (hydrocortisone, ascorbic acid, HEPES, Triton X-100 and bFGF), Gibco (Barcelona, Spain) (FBS, penicillin–streptomycin, chemically defined lipid concentrate, HBSS, collagen I rat protein and trypsin-EDTA), Lona (Barcelona, Spain) (EBM-2 medium).
Dulbecco’s modified eagle’s medium (DMEM) with a high content of glucose, L-glutamine, HEPES, MEM non-Essential aminoacid, penicillin−streptomycin, trypsin-EDTA, Hank’s balanced salt solution (HBSS) and fetal bovine serum (FBS) for the cell culture of MDCK and MDCK-MDR1 cell lines were purchased from Sigma-Aldrich (Barcelona, Spain).
The products needed for the culture of hCMEC/D3 cells were purchased from Sigma-Aldrich (Barcelona, Spain) (hydrocortisone, ascorbic acid, HEPES, Triton X-100 and bFGF), Gibco (Barcelona, Spain) (FBS, penicillin–streptomycin, chemically defined lipid concentrate, HBSS, collagen I rat protein and trypsin-EDTA), Lona (Barcelona, Spain) (EBM-2 medium).
5
Influenza A Strain Characterization and Cell Line Engineering
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The influenza A strains A/Victoria/3/75 (VIC) and ΔNS1 [32] (link) were used throughout. Encephalo-myocarditis virus (EMCV) was used for IFN bioassay. The MDCK cell line was purchased from the ATCC and the A549 cell line [49] (link) was obtained from J.A. Melero. The generation of MDCK-V2 and A549/pr(IFN-β).GFP cells [50] (link)–[52] (link) has been described. The A549/pr(ISRE).Luc cells, stably expressing luciferase under an ISRE promoter were obtained from G. Adolf, Boehringer Ingelheim, Austria. They were further engineered to express BVDV/NPro (A549/pr(ISRE).Luc-BVDV-Npro) to render them IRF3-deficient and unable to generate IFN [53] (link). Antibodies used included monoclonal antibodies specific for β-actin (Sigma) and phospho-Akt (Ser473; Cell Signaling Technology) as well as polyclonal antibodies to ISG56 (Santa Cruz), MxA (Santa Cruz), phospho-IRF3 (Ser396; Cell Signaling Technology), Akt (pan; Cell Signaling Technology), cleaved Caspase-3 (Asp175; Cell Signaling Technology) and Stat1 (Cell Signaling Technology). The specific anti-NS1 and anti-NP rabbit antibodies [54] (link), [55] (link), anti-M1 mouse antibodies [56] (link), anti-NS1 rat antibodies [35] (link) and anti-V2 antibodies [51] (link) have been described previously.
6
Characterization of Drug Permeation Profiles
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The drugs that were tested (letrozole, gemcitabine, methotrexate and ganciclovir) were purchased from Sigma-Aldrich. The MDCK cell line was purchased from ATCC (Manassas, VA, USA), the MDCK-MDR1 cells were provided by Dr. Gottessman, MM (National Institute of Health, Bethesda, MD, USA) and the hCMEC/D3 and the U87-MG cell lines were given by Dr. Sarmento (i3S, Porto). The molecular properties of the drugs that were tested are summarized in Table 1 .
The following products were purchased from Sigma-Aldrich: Dulbecco’s Phosphate Buffered Saline (PBS), Dulbecco’s Modified Eagle’s Medium (DMEM), Hank’s balances salt solution (HBSS), Non-essential Amino Acid Solution, HEPES solution, Penicillin–Streptomycin, dimethylsulfoxide (DMSO), basic Fibroblast Growth Factor (bFGF), glutamine, fetal bovine serum (FBS), hydrocortisone, ascorbic acid, trypsin, acid water, acetonitrile (ACN) and fluorescein (4kDa, 40kDa, 70kDa). The Dulbecco’s Modified Eagle’s Medium enriched with pyruvate (DMEM+pyruvate) and the Chemically Defined Lipid Concentrate (CD-LP) were ordered from ThermoFisher and the Endothelial Cell Growth Basal Medium (EBM-2), was bought from Lonza.
The following products were purchased from Sigma-Aldrich: Dulbecco’s Phosphate Buffered Saline (PBS), Dulbecco’s Modified Eagle’s Medium (DMEM), Hank’s balances salt solution (HBSS), Non-essential Amino Acid Solution, HEPES solution, Penicillin–Streptomycin, dimethylsulfoxide (DMSO), basic Fibroblast Growth Factor (bFGF), glutamine, fetal bovine serum (FBS), hydrocortisone, ascorbic acid, trypsin, acid water, acetonitrile (ACN) and fluorescein (4kDa, 40kDa, 70kDa). The Dulbecco’s Modified Eagle’s Medium enriched with pyruvate (DMEM+pyruvate) and the Chemically Defined Lipid Concentrate (CD-LP) were ordered from ThermoFisher and the Endothelial Cell Growth Basal Medium (EBM-2), was bought from Lonza.
7
Influenza and Cancer Cell Culture
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Influenza A and B viruses (strain A/Puerto Rico/8/1934 – H1N1, A/Victoria/3/75 – H3N2, B/Lee/40 and B/Maryland/1/59) and MDCK cell line were purchased from ATCC (Manassas, VA, USA). Lung cancer cell (NCL-H1299), human ovary cancer cells (HeLa and SKOV) purchased from ATCC (Manassas, VA, USA) and cervix cancer cell (CaSki) were from KCLB (Korean Cell line Bank, Seoul, South Korea). RPMI-1640, DMEM medium (Dulbecco’s Modified Eagle’s Medium), fetal bovine serum (FBS), antibiotic-antimytotic and trypsin-EDTA was supplied by Gibco BRL (Grand Island, NY, USA). The cell culture dish and 96 well plates were purchased from Falcon (BD Bioscience, Franklin Lakes, NJ, USA).
8
3D Collagen Gel Culture of MDCK Cells
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The MDCK cell line was obtained from American Type Culture Collection (ATCC, Manassas, VA) and cultured in MEM medium supplemented with 10% fetal bovine serum and 1% non-essential amino acid. The overlay 3-D culture was carried out as described previously with some modifications [12 (link)]. Briefly, 12-well culture plates were pre-coated evenly with 1.0 mg/mL pre-mixed collagen gel and then incubated at 37 °C for 30 min to allow the collagen gel to solidify. MDCK cells or MDCK cells with TAp73-KD, ΔNp73-KD, p21-KD or PUMA-KD (5,000 cells) suspended in 1.0 mL collagen gel mixture were seeded on the top of pre-gelled layer, and then incubated for 30 min at 37 °C to solidify. MEM growth medium was gently added to the top of each gel and incubated at 37 °C in a humidified 5% CO2. Culture medium was renewed every third day.
9
In Vitro and In Vivo Analysis of ASBT
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The Caco-2 cell line (human colorectal adenocarcinoma epithelial cells) and MDCK cell line (Madin–Darby canine kidney cells) were acquired from American Type Culture Collection (Manassas, VA, USA). The MDCK-ASBT cell line, which overexpresses ASBT, was transfected by a previously described method [45 (link)]. Complete culture DMEM medium was supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA), 1% penicillin–streptomycin (Thermo Fisher Scientific), and 1% 1 × non-essential amino acid (Thermo Fisher Scientific). The cell culture environment was kept at 37 ℃ with 5% carbon dioxide and humidity, and the culture medium (DMEM) was refreshed every 2–3 days. When the cell confluence reached 80%, the cells were subcultured onto either a Transwell plate (Corning Inc.) for the permeability assay or a six-well plate (Corning Inc.) for the western blot.
The Institutional Animal Care and Use Committee guideline of Seoul National University approved all protocols for in vivo experiments involving animals (SNU-130801–3-1). Sprague–Dawley rats (male, 6 weeks old) and C57BLKs/J db/db mice (male, 7 weeks old) were purchased from Orient Bio Inc. (Seongnam-si, Republic of Korea) and Japan SLC, Inc. (Shizuoka, Japan), respectively. Prior to their use, all animals were stabilized for 1 week under controlled conditions (23 ℃ ± 2 ℃, 55% ± 10% humidity).
The Institutional Animal Care and Use Committee guideline of Seoul National University approved all protocols for in vivo experiments involving animals (SNU-130801–3-1). Sprague–Dawley rats (male, 6 weeks old) and C57BLKs/J db/db mice (male, 7 weeks old) were purchased from Orient Bio Inc. (Seongnam-si, Republic of Korea) and Japan SLC, Inc. (Shizuoka, Japan), respectively. Prior to their use, all animals were stabilized for 1 week under controlled conditions (23 ℃ ± 2 ℃, 55% ± 10% humidity).
10
Cell Line Characterization and Validation
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Human breast (H1500, MCF7, T47D, ZR75-1, MDA-231, and MDA-157), colon (HCT116, DLD-1, HCT15, and SW480), and lung (A549) cancer cell lines, mouse NMuMG, 4T1 and NIH3T3 cell lines, and the MDCK cell line were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and cultured in the appropriate medium with supplements as recommended by ATCC. The SUM149 human breast cancer cell line was obtained from Asterand (Detroit, MI) and cultured in Ham’s F-12 supplemented with 5% fetal bovine serum (FBS), 5 µg ml−1 insulin, and 1 µg ml−1 hydrocortisone. The IMR-90 and TIG-1 normal human fetal lung fibroblasts were obtained from Coriell Institute for Medical Research (Camden, NJ) and cultured in Eagle’s minimum essential medium supplemented with 15% FBS. All cell lines were tested for mycoplasma contamination via PCR (e-Myco Plus kit; iNtRON Biotechnology, Kirkland, WA) and were found to be negative. In addition, all cell lines are routinely checked for morphologic and growth changes, to probe for cross-contamination or genetically drift. If present, cell lines were re-authenticated using the short tandem repeat profiling service by ATCC.
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