Following informed and written consent, human URECs (hURECs) were isolated from urine collected from two JBTS CEP290 patients and two unrelated wild-type controls. One JBTS patient carried in homozygosis the common CEP290 variant NM_025114.4; c.5668G>T; p.G1890X, whereas the second JBTS patient was compound het for the CEP290 variants c.5668G>T; p.G1890X and c.2495_2512delInATCT; p.T832Nfs*12. hURECs were cultured as described previously (Molinari et al., 2020 (link)). Briefly, cells were isolated from urine samples through repeated centrifugation passages and kept for the first 96 h in Dulbecco's medium Eagle medium/high glucose and Ham's F12 nutrient mix (1:1), supplemented with 10% (v/v) fetal bovine serum (FBS), 140 U/ml penicillin, 140 μg/ml streptomycin, 3.5 μg/ml amphotericin B and REGM SingleQuot kit supplements (Lonza), at 37°C in a humidified atmosphere of 5% (v/v) CO2. After isolation for 96 h, medium was changed to Renal Epithelial Cell Growth Basal Medium (REBM, Lonza) supplemented with 0.5% FBS, 140 U/ml penicillin, 140 μg/ml streptomycin, 3.5 μg/ml amphotericin B and REGM SingleQuot kit supplements (Lonza). Before collection, cells were cultured in the same medium devoid of FBS for 48 h to induce ciliogenesis.
Amphotericin b
Manufactured by Lonza
Sourced in United States, Switzerland, Belgium, United Kingdom, France
Amphotericin B is a laboratory product manufactured by Lonza. It is a naturally occurring antifungal compound commonly used in research and development applications.
Automatically generated - may contain errors
Lab products found in correlation
Streptomycin, by Lonza (54 mentions)
Penicillin, by Lonza (52 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (19 mentions)
Gentamicin, by Lonza (17 mentions)
Hydrocortisone, by Lonza (13 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (12 mentions)
Penicillin streptomycin, by Lonza (11 mentions)
Fetal bovine serum (fbs), by Lonza (11 mentions)
Ascorbic acid, by Lonza (9 mentions)
Hepes, by Lonza (8 mentions)
L glutamine, by Lonza (8 mentions)
Streptomycin, by Thermo Fisher Scientific (8 mentions)
Penicillin, by Thermo Fisher Scientific (8 mentions)
Heparin, by Lonza (6 mentions)
Rpmi 1640 medium, by Lonza (6 mentions)
Gentamicin sulfate, by Lonza (6 mentions)
Streptomycin sulfate, by Lonza (6 mentions)
L glutamine, by Thermo Fisher Scientific (6 mentions)
Penicillin, by Merck Group (6 mentions)
Human epidermal growth factor (hegf), by Lonza (6 mentions)
130 protocols using amphotericin b
1
Isolation and culture of human UREC
2
Cytotoxicity Evaluation of Peptides
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The human keratinocytes were maintained in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum (FBS; Hyclone, USA), penicillin G (100 U/mL), and streptomycin (100 μg/mL) (Invitrogen, USA). Human umbilical vein endothelial cells (HUVECs) were grown on 0.1% gelatin-coated cell culture dishes in M199 medium (Welgene, Korea) supplemented with 20% (v/v) FBS, 3 ng/mL basic fibroblast growth factor (R&D Systems, USA), 5 U/mL of heparin (Sigma, USA), and a penicillin-streptomycin-amphotericin B mixture (100 U/mL potassium penicillin, 100 mg/mL streptomycin sulfate, and 250 ng/mL amphotericin B; Lonza, Belgium) as a complete medium. Cells were cultured at 37°C in a humidified incubator with 5% CO2. Cells were plated in 96-well tissue culture plates (2 × 104 cells per well). After 1 day, they were treated with various concentrations (25, 50, 100, and 200 μg/mL) of peptides. Melittin (Sigma, USA) was used as the positive control. After incubation for 24 h, the viability of the cells was assessed by the Cell Titer 96 AQueous One Solution Cell Proliferation Assay according to the manufacturer’s protocol (Promega, USA). The optical density at 490 nm was measured with a microplate reader (Beckman DTX 8800 Multi Detector, USA).
3
Toluene Exposure and Cell Culture
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Toluene [C6H5CH3]: anhydrous colorless liquid substance with a pungent aromatic odor, molecular weight 92.13 g/mol, purity of 95%, CAS No. was 108-88-3, was purchased from El-Gomhouria Company for pharmaceutical, Egypt. 1 gm. toluene fluid was dissolved in 100 gm. olive oil to prepare 1% Tol solution, 1% Tol was the commonest dose to which industrial workers exposed to16 (link). Dulbecco’s Modified Eagle Medium (DMEM): 4.5 g/L glucose, with L-glutamine (Cat no. 12-604Q) was purchased from Lonza Bioproducts Walkersville, MD 21,793–0127 USA. Penicillin–Streptomycin-Amphotericin B mixture: 10,000 units/ml Potassium Penicillin, 10,000 µg/mL Streptomycin Sulfate and 25 µg/ml Amphotericin B cat no. 17-745E used as 10 ml/L from Lonza Bioproducts Walkersville, MD 21793-0127 USA. Trypsin/EDTA solution: 0.25% trypsin containing 0.02% EDTA cat no. CC-5012from Lonza Bioproducts Walkersville, MD 21793-0127, USA.
4
Isolation and Culture of Human Endothelial and Renal Cells
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Human umbilical vein endothelial cells (HUVEC) were purchased from BD Biosciences (Franklin Lakes, NJ) and cultured in EGM-2 media with EBM-2 supplements without gentamycin or amphotericin B (Lonza, Basel, Switzerland). Adult renal fibroblasts were purchased from DV Biologics (Yorba Linda, CA) and grown in Fibroblast Cellutions Medium with Fibroblast Cellutions supplement (DV Biologics, Yorba Linda, CA). Primary human RPTEC were purchased from four different commercial vendors (Lonza lot number 0000385391), Sciencell (lot number 11022; Carlsbad, CA), Zen-Bio (lot number RPCT082011; Research Triangle Park, NC), Lifeline Cell Technology (lot number 02685; Frederick, MD) and cultured according to the manufacturer's instructions.
5
Osteogenic Differentiation of BM-hMSCs
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BM-hMSCs were isolated as previously described17 (link). For this study we have used mainly cells at passage 3. For proliferation, cells were cultured at 37 °C in a humidified incubator with 5% CO2 in maintenance medium (MM), low-glucose DMEM (Dulbecco’s modified Eagle’s) supplemented with 10% FBS, 1% glutamine, 50 μg/ml penicillin-streptomycin and amphotericin B (Lonza Group Ltd.). To induce osteogenesis, BM-hMSCs were maintained in Osteogenic Differentiating Medium (ODM), α-MEM (Minimum Essential Medium) supplemented with 10% FBS, antibiotics and the osteogenic mixture containing 100nM dexamethasone, 5 mM β-glycerophosphate disodium and 50 mg/ml ascorbic acid (Sigma-Aldrich, S. Louis, MO, USA). Treatment lasted up to 27 days and the medium was changed every 3 days. The study was conducted in accordance with the Review Board of Fondazione IRCCS Policlinico San Matteo and the University of Pavia (2011).
6
Isolation and Culture of CD4+ T-cells
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Peripheral blood samples of healthy donors were provided by the Austrian Red Cross (Vienna, Austria) upon informed written consent. Peripheral blood mononuclear cells (PBMC) were isolated by standard Ficoll‐Paque centrifugation. CD4+ T‐cells were isolated using MagniSort Human CD4 T‐cell Enrichment Kit (Invitrogen/Thermo Fisher Scientific, Waltham, USA) according to manufacturing instructions. Purity was assessed by flow cytometric analyses and found to be above 95% for CD4+ T‐cells. All functional assays were performed in IMDM (Gibco, Thermo Fisher Scientific) supplemented with 10% of fetal calf serum (Gibco), 10 µg/mL of gentamycin (Gibco), and 1.25 µg/mL of amphotericin B (Lonza, Walkersville, MD, USA).
7
Primary HIFs Isolation and Culture
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Primary HIFs were isolated and cultured with some modifications as previously described [39 (link)]. Briefly, HIFs were derived from outgrowths of minced colonic mucosa explants placed on etched polystyrene flasks containing HIFs growth medium consisting of Dulbecco’s modified Eagle’s medium/high glucose (Hyclone, Logan, UT), 10% fetal bovine serum (American Type Culture Collection, Manassas, VA), 4 mmol/L L-glutamine (Gibco, Carlsbad, CA), 25 mmol/L HEPES, 100 U/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B (all purchased from Lonza, Walkersville, MD) and used between passage 6 and 10 at 80% confluence. HIFs were isolated from normal colon segments of patients undergoing resection due to colorectal cancer. All normal colon segments were macroscopically confirmed by a pathologist after surgical resection. The project was performed in accordance with the guidelines of the Institutional Review Board of the CHA Bundang Medical Center.
8
Cytotoxic Evaluation of Hydroxytyrosylalkylether Derivatives
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Dulbecco's Modified Eagle Medium (DMEM) 1g/L glucose, penicillin/streptomycin, amphotericin B, L-glutamine and bovine foetal serum (FBS) were supplied by Biowhittaker-Lonza (Walkersville, MD, USA).
The general reagents used for the different assays were obtained from Sigma-Aldrich (Merck; Darmstadt, Germany). Caspase-Glo® 3/7 Assay kit was purchased from Promega Biotech Ibérica (Madrid, Spain) and Matrigel was from Corning (New York, NY, USA).
Rabbit anti-PARP antibody was from Cell Signaling Techonology (Denver, MA, USA) and detects the 116 kDa full length from PARP and the 89 kDa cleaved fragment; rabbit anti-cleaved-lamin A antibody was from Cell Signaling Technology and detects the small cleavage fragment of lamin A (28 kDa). Rabbit anti-GAPDH antibody was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The HRP-linked goat anti-rabbit IgG antibody was purchased from Sigma-Aldrich (Merck, Darmstadt, Germany).
The hydroxytyrosylalkylether derivatives HT C1, C2, C4, C6, C8 and C12 (Figure 1A) tested in this study were synthesized as previously described (Madrona et al., 2009; (link)Pereira-Caro et al., 2011) (link). The compounds were dissolved in dimethyl sulfoxide (DMSO) at a stock concentration of 200 mM and stored at -20ºC until use.
The general reagents used for the different assays were obtained from Sigma-Aldrich (Merck; Darmstadt, Germany). Caspase-Glo® 3/7 Assay kit was purchased from Promega Biotech Ibérica (Madrid, Spain) and Matrigel was from Corning (New York, NY, USA).
Rabbit anti-PARP antibody was from Cell Signaling Techonology (Denver, MA, USA) and detects the 116 kDa full length from PARP and the 89 kDa cleaved fragment; rabbit anti-cleaved-lamin A antibody was from Cell Signaling Technology and detects the small cleavage fragment of lamin A (28 kDa). Rabbit anti-GAPDH antibody was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The HRP-linked goat anti-rabbit IgG antibody was purchased from Sigma-Aldrich (Merck, Darmstadt, Germany).
The hydroxytyrosylalkylether derivatives HT C1, C2, C4, C6, C8 and C12 (Figure 1A) tested in this study were synthesized as previously described (Madrona et al., 2009; (link)Pereira-Caro et al., 2011) (link). The compounds were dissolved in dimethyl sulfoxide (DMSO) at a stock concentration of 200 mM and stored at -20ºC until use.
9
Isolation and Culture of Primary Human Intestinal Myofibroblasts
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Primary HIMFs were isolated and cultured as per a previously described protocol with some modifications [36 (link)]. Briefly, HIMFs were derived from the outgrowths of minced colonic mucosa explants placed on etched polystyrene flasks containing HIMFs growth medium consisting of Dulbecco’s modified Eagle’s medium/high glucose (Hyclone, Logan, UT), 10% FBS (American Type Culture Collection, Manassas, VA), 4 mmol/L L-glutamine (Gibco, Carlsbad, CA), 25 mmol/L HEPES, 100 U/mL penicillin, 100 μg/mL streptomycin, and 0.25 μg/mL amphotericin B (all purchased from Lonza, Walkersville, MD) and used between passages 6 and 10 at 80% confluence. HIMFs were isolated from normal colon segments of patients undergoing resection for colorectal cancer. Following surgical resection, a pathologist removed the segments of the colon that were grossly normal from the area near the proximal resection margin. The periphery of the normal colon segments, which was used for isolation, was histologically confirmed under a microscope. The project was performed in accordance with the guidelines of the Institutional Review Board of the CHA Bundang Medical Center.
10
Evaluation of Sorafenib, Bevacizumab, and Fucoidan in HUH-7 Cells
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Human hepatocellular carcinoma cell line HUH-7 was purchased from Vacsera (Giza, Egypt), sorafenib p- Toluenesulfonate salt was purchased from LC Laboratories (Woburn, MA, United States), Avastin® (bevacizumab, Genentech, United States) was purchased, in its formulated commercial preparation, from a community Pharmacy (Cairo, Egypt) and fucoidan extracted from Laminaria Japonica was purchased as a crude extract from Buchem BV (Holland). Dulbecco’s modified Eagle’s medium (DMEM) medium and fetal bovine serum (FBS) were purchased from Gibco®, Thermo Fisher Scientific, United States. Antibiotic-antimycotic mixture (100 U/ml of penicillin, 0.1 U/ml streptomycin and 0.25 μg/ml of Amphotericin B) was purchased from Lonza®, Walkersville, MD, United States. All other chemicals were purchased from Sigma Aldrich, United States unless otherwise specified.
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