Primary cultures were performed as previously described (Fortin et al., 2013 (link)). Briefly, pituitaries were collected from 10 week old Bmpr1afl/fl;Acvr1fl/fl male and female mice in M199 medium supplemented with 10% (v/v) fetal bovine serum (FBS). Pituitaries were washed three times in Hank’s Balanced Salt Solution (HBSS) with 150 µmol l−1 of CaCl2, cut several times with a scalpel, and digested in collagenase (1.5 mg/ml) (Sigma #C-0130; diluted in HBSS with 30 mg/ml BSA, pH 7.4, 40 µl/pituitary) at 37°C for 2 hours. The suspension was then washed with 5 ml calcium-free HBSS, centrifuged for 5 min at 1000 × g, and resuspended in pancreatin solution (Sigma P3292; 4.5 mg/ml in calcium-free HBSS; 40 µl/pituitary). Pancreatin digestion was performed in a 37°C water bath with manual agitation for 8 to 10 min. Finally, the cell suspension was washed three times in 5 ml M199 media containing 10% FBS and cells were seeded at density of 5 × 105/well in 48-well plates.
P3292
Manufactured by Merck Group
Sourced in Italy, United States
The P3292 is a laboratory equipment product from Merck Group. It is a device designed for general laboratory use. The core function of the P3292 is to provide a controlled and precise environment for various scientific experiments and analyses. Further details about the intended use or specific features of this product are not available.
Automatically generated - may contain errors
Lab products found in correlation
P6887, by Merck Group (5 mentions)
Cellulose acetate membrane filter, by Merck Group (2 mentions)
C0130, by Merck Group (1 mentions)
Medium 199, by Merck Group (1 mentions)
Wistar rat, by Janvier Labs (1 mentions)
P4937, by Merck Group (1 mentions)
Ls004174, by Worthington (1 mentions)
Trichloroacetic acid, by Merck Group (1 mentions)
Sodium hydroxide (naoh), by Merck Group (1 mentions)
8 protocols using p3292
1
Pituitary Cell Culture from Transgenic Mice
2
Isolation and culture of rat cardiomyocytes
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NCM and NCF were isolated from heart ventricles of one- to three-day old Wistar rats (Janvier Labs, Le Genest-Saint-Isle, France) as previously described [41 (link)]. Briefly, minced ventricular tissue was digested with type II collagenase (LS004174, Worthington, Lakewood, NJ, USA) and pancreatin (P3292, Sigma-Aldrich, St. Louis, MO, USA), and dissociated cells were pelleted by centrifugation at 800× g. NCMs were separated from NCFs via discontinuous Percoll gradient (bottom 58.5%, top 40.5% [v/v], P4937, Sigma-Aldrich, St. Louis, MO, USA) for 30 min at 1600× g. NCMs were seeded at 2.5 × 106 cells per 100 mm collagen-coated culture dish and were cultured in a 4:1 mixture of DMEM (D1152, Sigma Aldrich, St. Louis, MO, USA) and Medium 199 (M4530 Sigma-Aldrich, St. Louis, MO, USA), supplemented with 10% horse serum (HS) (16050122, Thermo Fisher Scientific, Waltham, MA, USA), 5% fetal calf serum (FCS) (30-2020, ATCC, Manassas, VA, USA), and 1% penicillin-streptomycin (PS) (15140-122, Gibco, Waltham, MA, USA) for 5 days at 37 °C under 5% CO2 atmosphere. NCFs were seeded at 2 × 106 cells per 100 mm culture dish and were cultured in DMEM (31966-02, Gibco, Waltham, MA, USA), 10% FCS, and 1% PS for 48 h at 37 °C under 5% CO2 atmosphere. NCF at passage 1 were used for experiments.
3
In Vitro Gastrointestinal Digestion of Food Samples
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The in vitro gastrointestinal digestion of honey, cheese, and C-H samples was simulated using pepsin and pancreatin according to the method reported by Simonetti, Gambacorta, and Perna (2016) , with some modifications. Fifteen grams of each sample were mixed with 40 mL of bidistilled water and homogenized in a Stomacher (Steward Stomacher 400 Lab Blender, London, UK) for 1 min for simulate the human chewing. Then HCl (3 mol/L; Sigma-Aldrich, Milan, Italy) was used to bring the pH of the solution to 2 (model PHM 92, Radiometer, Copenhagen, Denmark), and stomach phase was simulated by adding pepsin (Sigma-Aldrich P6887, Milan, Italy) at a 1:200 (enzyme:substrate) ratio. After 2 h of digestion at 37 °C, the pepsin was inactivated by adjusting the pH to 7.2 with 1 mol/L NaHCO 3 and the pancreatin (Sigma-Aldrich P3292, Milan, Italy) was added at a 1:25 (enzyme:substrate) ratio to simulated intestinal phase. After 4 h of digestion at 37 °C, pancreatin activity in the solution was terminated by heating for 10 min at 95 °C. Samples were collected before adding the enzymes (undigested sample), and after in vitro digestion (digested sample). Each sample was centrifuged at 5,000×g for 20 min at 4 °C to remove large particles, the supernatant was filtered through a 0.2 μm cellulose acetate membrane filter (Sigma-Aldrich), and it was frozen and kept a -55 °C until analysis.
4
Simulated Gastrointestinal Digestion of Fortified Yogurts
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In vitro gastrointestinal digestions
The in vitro gastrointestinal digestion of fortified yogurts was simulated according to the method reported by Simonetti et al. (2016) (link), with some modifications. Briefly, 8 g of each sample were mixed with 20 mL of bidistilled water and homogenized in a Stomacher (Steward Stomacher 400 Lab Blender, London, UK) for 1 min to simulate the human chewing. Then 3 M HCl was used to bring the solution pH to 2 (model PHM 92, Radiometer, Copenhagen, Denmark), and stomach phase was simulated by adding 20 mL gastric juice (1.25 mg pepsin per mL of 0.1 M HCl; Sigma-Aldrich P6887, Milan, Ialy). After 2 hr of digestion at 37 • C, the pepsin was inactivated by adjusting the pH to 7.2 with 1 M NaHCO 3 and 40 mL pancreatin juice (4.50 mg pancreatin per mL of 20 mM phosphate buffer, pH 7.2; Sigma-Aldrich P3292) was added to simulate intestinal phase. After 4 hr of digestion, pancreatin activity was terminated by heating for 10 min at 95 • C. Aliquots of the samples were collected before adding the enzymes (undigested), after peptic (gastric) and pancreatic (intestinal) digestion. The digestion was carried out in triplicate.
The in vitro gastrointestinal digestion of fortified yogurts was simulated according to the method reported by Simonetti et al. (2016) (link), with some modifications. Briefly, 8 g of each sample were mixed with 20 mL of bidistilled water and homogenized in a Stomacher (Steward Stomacher 400 Lab Blender, London, UK) for 1 min to simulate the human chewing. Then 3 M HCl was used to bring the solution pH to 2 (model PHM 92, Radiometer, Copenhagen, Denmark), and stomach phase was simulated by adding 20 mL gastric juice (1.25 mg pepsin per mL of 0.1 M HCl; Sigma-Aldrich P6887, Milan, Ialy). After 2 hr of digestion at 37 • C, the pepsin was inactivated by adjusting the pH to 7.2 with 1 M NaHCO 3 and 40 mL pancreatin juice (4.50 mg pancreatin per mL of 20 mM phosphate buffer, pH 7.2; Sigma-Aldrich P3292) was added to simulate intestinal phase. After 4 hr of digestion, pancreatin activity was terminated by heating for 10 min at 95 • C. Aliquots of the samples were collected before adding the enzymes (undigested), after peptic (gastric) and pancreatic (intestinal) digestion. The digestion was carried out in triplicate.
5
In Vitro Digestion of Cooked Meat Proteins
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The in vitro gastrointestinal digestion of cooked meat proteins was simulated using pepsin and pancreatin according to the method of Laparra, Vélez, Montoro, Barberá, and Farré (2003) , with some modifications. Five gram of cooked meat was mixed with 50 mL of bidistilled water and human chewing was simulated by using Stomacher (Steward Stomacher 400 Lab Blender, London, UK) for 1 min. After that, the pH was adjusted to 2 (model PHM 92, Radiometer, Copenhagen, Denmark) with 3 M HCl (Sigma-Aldrich, Milan, Italy) and stomach phase was simulated by adding pepsin (Sigma-Aldrich P6887, Milan, Italy) at a 1:100 (enzyme:substrate) ratio. After 2 h of digestion at 37 °C and continuous stirring, the enzyme was inactivated by adjusting the pH to 7.2 with 1 M NaHCO 3 . Then, pancreatin (Sigma-Aldrich P3292, Milan, Italy) was added at a 1:50 (enzyme:substrate) ratio to simulated intestinal phase. After 3 h of digestion at 37 °C, enzyme activity was terminated by heating for 10 min at 95 °C. The reaction mixture was centrifuged at 5000g for 20 min at 4 °C (CR 412, Jouan, Saint-Herblain, France) to remove large particles, the supernatant was filtered through a 0.45 lm cellulose acetate membrane filter (Sigma-Aldrich, Milan, Italy), and it was frozen and kept a À55 °C until analysis.
6
In Vitro Gastrointestinal Digestion of Food Samples
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The in vitro gastrointestinal digestion of honey, cheese, and C-H samples was simulated using pepsin and pancreatin according to the method reported by Simonetti, Gambacorta, and Perna (2016) , with some modifications. Fifteen grams of each sample were mixed with 40 mL of bidistilled water and homogenized in a Stomacher (Steward Stomacher 400 Lab Blender, London, UK) for 1 min for simulate the human chewing. Then HCl (3 mol/L; Sigma-Aldrich, Milan, Italy) was used to bring the pH of the solution to 2 (model PHM 92, Radiometer, Copenhagen, Denmark), and stomach phase was simulated by adding pepsin (Sigma-Aldrich P6887, Milan, Italy) at a 1:200 (enzyme:substrate) ratio. After 2 h of digestion at 37 °C, the pepsin was inactivated by adjusting the pH to 7.2 with 1 mol/L NaHCO 3 and the pancreatin (Sigma-Aldrich P3292, Milan, Italy) was added at a 1:25 (enzyme:substrate) ratio to simulated intestinal phase. After 4 h of digestion at 37 °C, pancreatin activity in the solution was terminated by heating for 10 min at 95 °C. Samples were collected before adding the enzymes (undigested sample), and after in vitro digestion (digested sample). Each sample was centrifuged at 5,000×g for 20 min at 4 °C to remove large particles, the supernatant was filtered through a 0.2 μm cellulose acetate membrane filter (Sigma-Aldrich), and it was frozen and kept a -55 °C until analysis.
7
In Vitro Intestinal Digestion Model
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The simulation of intestinal digestion consisted of a jacketed glass reactor (1 L capacity) maintained at 37 °C with continuous magnetic stirring at 120 rpm in a temperature-controlled circulating water bath throughout the test.
The in vitro digestions were carried out according to the method of Sozer and others (2014), with modifications. 4 g of ground sample, 100 mL 0.05 mol/L of sodium potassium phosphate buffer (pH 6.9) and 5 mL of 2.5g/100 mL pancreatin (P3292, pancreatin from porcine pancreas containing trypsin, amylase, lipase, ribonuclease and protease, Sigma-Aldrich, St. Louis, Mo., U.S.A) in 0.1 mol/L of pH 6 maleate buffer were placed in the jacketed glass reactor and maintained there for 120 min. Aliquots (7.5 mL) were removed at 20, 60, 90 and 120 min, placed in boiling water for 5 min, and cooled on ice. They were then centrifuged at 6600 rpm for 5 min and the supernatant was analyzed for reducing sugar content using the dinitrosalicylic acid (DNS) colorimetric method. The amount of digested starch was determined by multiplying the reducing sugar values by a 0.9 stoichiometric conversion constant for glucose to starch (Hardacre and others 2015) . For each formulation, two different sponge cakes were analyzed.
The in vitro digestions were carried out according to the method of Sozer and others (2014), with modifications. 4 g of ground sample, 100 mL 0.05 mol/L of sodium potassium phosphate buffer (pH 6.9) and 5 mL of 2.5g/100 mL pancreatin (P3292, pancreatin from porcine pancreas containing trypsin, amylase, lipase, ribonuclease and protease, Sigma-Aldrich, St. Louis, Mo., U.S.A) in 0.1 mol/L of pH 6 maleate buffer were placed in the jacketed glass reactor and maintained there for 120 min. Aliquots (7.5 mL) were removed at 20, 60, 90 and 120 min, placed in boiling water for 5 min, and cooled on ice. They were then centrifuged at 6600 rpm for 5 min and the supernatant was analyzed for reducing sugar content using the dinitrosalicylic acid (DNS) colorimetric method. The amount of digested starch was determined by multiplying the reducing sugar values by a 0.9 stoichiometric conversion constant for glucose to starch (Hardacre and others 2015) . For each formulation, two different sponge cakes were analyzed.
8
Carbohydrate, Protein, and Lipid Digestibility
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To assess in vitro carbohydrate digestibility, rapidly (RDS) and slowly (SDS) digestible starch fractions were measured as reported by Marti et al. (2017) and expressed as relative percentage (%). Procedure by the same Authors were used to quantify RS, expressed as a percentage of total starch (g/100 g TS). For each sample, 6 analytical replicates were performed.
For in vitro protein and lipid digestibility, pepsin from porcine gastric mucosa (P6887, Sigma-Aldrich, Saint Louis, MO, USA) and pancreatin from porcine pancreas cell (P3292, Sigma-Aldrich, Saint Louis, MO, USA) were used. In vitro protein digestibility was measured in quadruplicate, following the two-step procedure by Zhou et al. (2017) .
Undigested peptides (molecular weight > 3000 Da) were evaluated after precipitation with trichloroacetic acid (Sigma-Aldrich, Saint Louis, MO, USA; 10 g/100 mL final concentration), resuspension in NaOH (Sigma-Aldrich, Saint Louis, MO, USA; 2 mol/L) and quantification by Lowry assay (Lowry, Rosebrough, Farr, & Randall, 1951) .
Protein digestibility was expressed as the amount of digested proteins on the amount of total protein present in the sample at the beginning of each step (g/100 g proteins).
For in vitro protein and lipid digestibility, pepsin from porcine gastric mucosa (P6887, Sigma-Aldrich, Saint Louis, MO, USA) and pancreatin from porcine pancreas cell (P3292, Sigma-Aldrich, Saint Louis, MO, USA) were used. In vitro protein digestibility was measured in quadruplicate, following the two-step procedure by Zhou et al. (2017) .
Undigested peptides (molecular weight > 3000 Da) were evaluated after precipitation with trichloroacetic acid (Sigma-Aldrich, Saint Louis, MO, USA; 10 g/100 mL final concentration), resuspension in NaOH (Sigma-Aldrich, Saint Louis, MO, USA; 2 mol/L) and quantification by Lowry assay (Lowry, Rosebrough, Farr, & Randall, 1951) .
Protein digestibility was expressed as the amount of digested proteins on the amount of total protein present in the sample at the beginning of each step (g/100 g proteins).
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