The SGC7901 gastric cancer cell line was plated on 96-well plates at a density of 2×103 cells/well. The cells were transiently transfected with the above five luciferase reporter plasmids using Lipofectamine Plus system according to the manufacturer’s instructions (Invitrogen Life Technologies, Carlsbad, CA, USA). To evaluate the effect of gastrin (Sigma-Aldrich), the transfected cells were incubated with gastrin (1×10−7 mol/l) for 24 h after 48 h transfection. The luciferase activity of the transfected cells without or with gastrin incubation was measured by Luciferase Assay System (Promega Corporation, Madison, WI, USA) in a Glomax fluorescence detector (Promega Corporation) according to the manufacturer’s instructions. All assays were conducted in triplicate.
Gastrin
Manufactured by Merck Group
Sourced in United States, United Kingdom
Gastrin is a laboratory product manufactured by Merck Group. It is a polypeptide hormone primarily produced by G cells in the stomach and duodenum. Gastrin's core function is to stimulate the production and secretion of gastric acid by the parietal cells in the stomach.
Automatically generated - may contain errors
Lab products found in correlation
Nicotinamide, by Merck Group (50 mentions)
N acetylcysteine, by Merck Group (37 mentions)
A83 01, by Bio-Techne (36 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (34 mentions)
Glutamax, by Thermo Fisher Scientific (32 mentions)
Fgf10, by Thermo Fisher Scientific (20 mentions)
Sb202190, by Merck Group (17 mentions)
Hepes, by Thermo Fisher Scientific (17 mentions)
Y 27632, by Merck Group (16 mentions)
Advanced dmem f12, by Thermo Fisher Scientific (16 mentions)
Noggin, by Thermo Fisher Scientific (15 mentions)
Matrigel, by BD (13 mentions)
Hepatocyte growth factor (hgf), by Thermo Fisher Scientific (13 mentions)
B27 supplement, by Thermo Fisher Scientific (12 mentions)
Primocin, by InvivoGen (10 mentions)
Addmem f12, by Thermo Fisher Scientific (10 mentions)
Matrigel, by Corning (9 mentions)
N acetyl l cysteine, by Merck Group (9 mentions)
A83 01, by Merck Group (9 mentions)
Matrigel, by Thermo Fisher Scientific (9 mentions)
82 protocols using gastrin
1
Gastrin Effect on Gastric Cancer Cells
2
Apoptosis Assay for Gastrin and Clusterin
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Caspase Glo® 3/7 Assay (Promega Corporation, Fitchburg, WI) was performed according to manufacturer’s instructions. Apoptosis was induced by 48 hours of serum-starvation. AGS-GR cells were treated with gastrin (G9020, Sigma-Aldrich) 5 or 10 nM and combinations of goat anti-CLU (C-18, sc-6419, Santa Cruz Biotechnology Inc.) 8 μg/ml, non-immunized goat polyclonal IgG 8 μg/ml, recombinant sCLU (Clusterin Human HEK293, RD172034100, BioVendor) 200 nM and cisplatin (479306, Sigma-Aldrich) 10 μM.
TiterTACS In Situ Detection Kit—Colorimetric (4822-96-K, R&D Systems, Minneapolis, MN) allows terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining of cells in a 96-well format, and was performed according to manufacturer’s instructions. Apoptosis was induced by 72 hours of serum-starvation. AGS-GR cells were treated with gastrin (G9020, Sigma-Aldrich) 10 nM and/or recombinant sCLU (Clusterin Human HEK293, RD172034100, BioVendor) 200 nM.
TiterTACS In Situ Detection Kit—Colorimetric (4822-96-K, R&D Systems, Minneapolis, MN) allows terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining of cells in a 96-well format, and was performed according to manufacturer’s instructions. Apoptosis was induced by 72 hours of serum-starvation. AGS-GR cells were treated with gastrin (G9020, Sigma-Aldrich) 10 nM and/or recombinant sCLU (Clusterin Human HEK293, RD172034100, BioVendor) 200 nM.
3
Pancreatic Organoid Culture Protocol
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Pancreatic organoids were derived and cultured as previously described20 (link),45 (link),46 (link). Briefly, adult pancreatic tissue was digested for 45–60 minutes at 37 °C with the collagenase/dispase dissociation Medium (1% in DMEM media (Gibco), supplemented with 1% FBS (Gibco) and Collagenase type XI 0.012% (w/v) (Sigma), dispase 0.012% (w/v) (Gibco)). Single ducts were manually picked and seeded in Matrigel (BD Bioscience). Ducts were cultured in AdDMEM/F12 (Invitrogen) supplemented with N2 and B27 (Lifesciences), 1.25 mM N-Acetylcysteine (Sigma), 10 nM gastrin (Sigma) and the growth factors: 50 ng/ml EGF (Peprotech), 5% RSPO1-conditioned media, 5% Noggin-conditioned media (in-house prepared), 100 ng/ml FGF10 (Peprotech) and 10 mM Nicotinamide (Sigma). Differentiation medium consisted in removal of EGF, RSPO1-conditioned media and addition of the Wnt Inhibitor IWP-2 (0,25 μM, Stemgent47 (link)) for the last 2 days of induction, with or without doxycycline, as indicated.
4
Liver Organoid Differentiation and Functional Assay
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Liver organoids were seeded and kept 7–10 days under the liver medium explained above (EM, expansion medium) supplemented with BMP7 (25 ng/ml). Then, the cultures were split and seeded accordingly in this EM supplemented with BMP7 for at least 2–4 days. Then, medium was changed to the differentiation medium (DM): AdDMEM/F12 medium supplemented with 1% N2 and 1% B27 and containing EGF (50 ng/ml), gastrin (10 nM, Sigma), HGF (25 ng/ml), FGF19 (100 ng/ml), A8301 (500 nM), DAPT (10 uM), BMP7 (25 ng/ml), and dexamethasone (30 uM). Differentiation medium was changed every 2–3 for a period of 11–13 days.
To assess hepatocyte function, culture medium was collected 24 hr after the last medium change. Functional studies were performed in the collected supernatant or in whole organoids, as described in theExtended Experimental Procedures .
To assess hepatocyte function, culture medium was collected 24 hr after the last medium change. Functional studies were performed in the collected supernatant or in whole organoids, as described in the
5
Establishment of Colorectal Cancer Organoids
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The tumor organoids were isolated as previously described [10 (link)]. Briefly, cancer tissues were incubated with collagenase type II (Sigma-Aldrich, Louis, MO, USA), dispase type II (Roche Applied Science, Mannheim, Germany), and Y-27632 (BioVision, Mountain View, CA, USA) for 1 h at 37°C. Isolated cells were washed with PBS and centrifuged at 300 ×g for 3 min. The cells were then embedded in Matrigel (growth factor reduced, phenol red free; Corning, NY, USA) and seeded in 4-well plates, followed by the addition of the culture medium. The composition of the CRC organoid culture medium was 1 × B27 (Gibco, Grand Island, NY, USA), 1.25 mM N-acetyl cysteine (United States Pharmacopeia, Rockville, MD, USA), 50 ng/mL human epidermal growth factor (BioVision), 50 ng/mL human Noggin (Peprotech, Rocky Hill, NJ, USA), 10 nM gastrin (Sigma-Aldrich), 500 nM A83-01 (BioVision), and 100 mg/mL primocin (InvivoGen, San Diego, CA, USA). To prevent anoikis, 10 μM Y-27632 was added to the culture medium during the first 2-3 days. When organoids were >200 µm, they were passaged by pipetting using the Gentle Cell Dissociation Reagent (STEMCELL Technologies, Vancouver, Canada) according to the manufacturer's instructions.
6
Propagation of Gastric Organoids from ApcMutant Mice
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Isolated gastric tumors from Apc1638N mice were trypsinized for single-cell culture, and mixed with Matrigel (BD Biosciences, San Jose, CA), and plated in 48-well plates. After polymerization of Matrigel, gastric culture medium (Advanced DMEM/F12 supplemented with B-27 Supplement (50×), N2, 1 mM N-acetylcysteine (Invitrogen, 5823 Newton Dr, Carlsbad, CA), and gastrin (10 nM; Sigma-Aldrich) containing growth factors (50 ng/mL EGF, Peprotech, Rocky Hill, New Jersey; 1 mg/mL R-spondin1; 100 ng/mL Noggin, Peprotech; 100 ng/mL FGF10, Preprotech; and Wnt3A conditioned medium)) was overlain. For the first two days after seeding, the medium was also supplemented with 10 mM ROCK inhibitor Y-27632 (Sigma Aldrich) to avoid anoikis. Growth factors were added every second day, and the entire medium was changed every two days. For passage, gastric organoids were removed from the Matrigel, mechanically dissociated, and transferred to fresh Matrigel. Passage was performed every week with a 1:5–1:8 split ratio.
7
Colorectal Adenoma Organoid Culture
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Human adenoma biopsy samples were obtained from patients (40–70 years old) for research purpose under the approval of the Ethical Committee of Medical Research, Huashan Hospital of Fudan University (2018-182). Informed consent had been obtained from all subjects involved.
Human colorectal adenoma samples were minced into 3 mm3, then washed with PBS three times. The tissue fragments were digested by collagenase solution (collagenase 0.125 mg/mL, dispase 0.5 mg/mL, FBS 1% in DMEM medium) for 50 min at 37 °C. Isolated epithelium was mixed with Matrigel (Corning) and cultured as previously described [19 (link)]. Briefly, CA organoid culture medium was the basal medium supplemented with 500 ng/mL RSPO1 (R&D system, Minneapolis, MN, USA), 100 ng/mL Noggin (R&D), 50 ng/mL EGF (Invitrogen, Carlsbad, USA), 10 nM gastrin (Sigma-Aldrich, St Louis, MO, USA), 500 nM A83-01, and 10 μM SB202190. In order to determine the optimum culture medium with selectivity for CA organoids, we compared CA medium with or without 10 ng/mL Wnt3a. Culture medium was changed every other day. Within 1 week, organoids were passaged at a 1:3 ratio with TrypLE digestion.
Human colorectal adenoma samples were minced into 3 mm3, then washed with PBS three times. The tissue fragments were digested by collagenase solution (collagenase 0.125 mg/mL, dispase 0.5 mg/mL, FBS 1% in DMEM medium) for 50 min at 37 °C. Isolated epithelium was mixed with Matrigel (Corning) and cultured as previously described [19 (link)]. Briefly, CA organoid culture medium was the basal medium supplemented with 500 ng/mL RSPO1 (R&D system, Minneapolis, MN, USA), 100 ng/mL Noggin (R&D), 50 ng/mL EGF (Invitrogen, Carlsbad, USA), 10 nM gastrin (Sigma-Aldrich, St Louis, MO, USA), 500 nM A83-01, and 10 μM SB202190. In order to determine the optimum culture medium with selectivity for CA organoids, we compared CA medium with or without 10 ng/mL Wnt3a. Culture medium was changed every other day. Within 1 week, organoids were passaged at a 1:3 ratio with TrypLE digestion.
8
Generation and Passaging of Liver Organoids
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The protocol of generation of liver organoids has been described previously.[26 ] We generated iHep‐organoids by seeding 50 000 iHep cells mixed with Matrigel into a well of a 24 well plate. After Matrigel solidification, 500 µL organoids generation medium was added per well. Organoids generation medium: AdDMEM/F12 (Thermo Scientific), 0.5% Penicillin‐Streptomycin, 1% GlutaMAX, 10 × 10−3 m HEPES, 1% B27 minus vitamin A, 15% R‐spodin1‐conditioned medium, 3 × 10−6 m ChIR99021 (Sigma), 10 × 10−3 m nicotinamide (Sigma),10 × 10−9 m gastrin (Sigma), 50 ng mL−1 EGF (Peprotech), 20 ng mL−1 TGF‐α (Peprotech), 100 ng mL−1 FGF7 (Peprotech), 100 ng mL−1 FGF‐10 (Peprotech), 50 ng mL−1 HGF (Peprotech), 2 × 10−3 m A83‐01(Tocris), 10 × 10−6 m Y‐27632, 1 × 10−6 m dexamethasone (Sigma), 10 ng mL−1 Oncostatin M. During generation, the medium was changed every 3 days. After 14 days generation, organoids were mechanically fragmented with pipette tips blowing and separated by centrifugation for 200 g 10 min. The pellets was re‐seed into Matrigel with a split ratio of 1:4 into a well of a 24 well plate. Then, the medium was changed every 3 days and the organoids could passaged every 10 days with a split ratio of 1:4.
9
Liver Organoid Differentiation Protocol
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Liver organoids were cultured for 7 days in expansion medium (EM) supplemented with bone morphogenetic protein 7 (BMP7, 25 ng/ml) (MedChemExpress, USA). Medium was then changed into the differentiating medium (DM) which consisted of AdDMEM/F12 supplemented with 1% of both N2 and B27, 50 ng/ml EGF (Peprotech, UK), 10 nM gastrin (Sigma, USA), 25 ng/ml HGF (Peprotech, UK), 100 ng/ml FGF19 (Peprotech, UK), 500 nM A83-01 (MedChemExpress, USA), 10 μM DAPT (MedChemExpress, USA), 25 ng/ml BMP7 (MedChemExpress, USA), and 30 μM dexamethasone (MedChemExpress, USA). Differentiation medium was changed every 2–3 days for 13–15 days of culture. Functional analysis was performed in the collected supernatant (24 h after the last medium change) or in whole organoids.
10
Culturing Primary Tumor Organoids
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