To detect the apoptosis of A549 cells, flow cytometry assay was conducted according to the previously described method [15 ]. Cells were counted and cultured into six-well plates (ABI, 2 × 105 cells/well) in a humidified 5% CO2 incubator (Thermo) at 37 °C overnight. The cells were subsequently treated with different concentrations of HPD and different doses of X-ray. After the medium was discarded, the cells were digested with pancreatin (GIBCO), followed by treatment with fresh medium to deactivate pancreatin and centrifugation (1000 rpm, 6 min). The supernatant was discarded and the cells were washed once with PBS and resuspended in 1× binding buffer (BD Biosciences; 400 μL 1× binding buffer for the control group and 100 μL 1× binding buffer for other groups). A total of 100 μL of the above solution was transferred into flow tubes and treated with 5 μL of fluorescein isothiocyanate (FITC)-Annexin V (BD Biosciences) and 5 μL of propidium iodide (PI, BD Biosciences, 50 μg/mL) (the control group was divided into unstained, Annexin V-stained, PI-stained, and Annexin V + PI-stained groups). After being incubated (in the absence of light) for 15 min at room temperature, the cells were treated with 400 μL of 1× binding buffer (BD Biosciences) and analyzed with a flow cytometer (BD Biosciences).
Pancreatin
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany, China
Pancreatin is a digestive enzyme complex derived from porcine pancreas. It contains a mixture of enzymes, including amylase, lipase, and protease, which are involved in the breakdown of carbohydrates, fats, and proteins, respectively. Pancreatin is commonly used in laboratory settings for various applications requiring these enzymatic functions.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (23 mentions)
Co2 incubator, by Thermo Fisher Scientific (11 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (10 mentions)
Collagenase, by Thermo Fisher Scientific (8 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (8 mentions)
Penicillin, by Thermo Fisher Scientific (6 mentions)
Streptomycin, by Thermo Fisher Scientific (6 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (6 mentions)
Dmem f12, by Thermo Fisher Scientific (5 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (5 mentions)
Collagenase, by Worthington (4 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (4 mentions)
Pepsin, by Thermo Fisher Scientific (3 mentions)
Gallic acid, by Merck Group (3 mentions)
Collagenase 2, by Thermo Fisher Scientific (3 mentions)
Bile salt, by Merck Group (3 mentions)
Tnf α, by Shanghai Enzyme-linked (3 mentions)
Il 1β, by Shanghai Enzyme-linked (3 mentions)
Collagenase type 2, by Worthington (3 mentions)
Rpmi 1640, by Thermo Fisher Scientific (3 mentions)
81 protocols using pancreatin
1
Detecting A549 Cell Apoptosis by Flow Cytometry
2
Microglia Culture and Activation Assay
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Primary cultures of microglia from neonatal C57BL/6J mice brain (P0-P3 neonates) were prepared as described previously [30] . Brie y, the entire brain region was dissociated into a single-cell suspension using 0.25% pancreatin (Gibco, California, USA). Neonates were decapitated under sterile conditions, cut off the scalp and skull, removed the brain tissue. And brain tissue was placed in a dish containing cold D-Hank's solution, pH 7.2, without calcium or magnesium (Gibco, California, USA, Cat# C14175500BT).
Tissues were enzymatically dissociated into a single-cell suspension using 0.25% pancreatin (Gibco, California, USA), then the mixed glial cells were cultured for 1 week in DMEM/F12 (Gibco, California, USA, Cat# C11330500BT) containing 10% fetal bovine serum (Gibco, California, USA) at 37 °C and 5% CO 2 .
The isolated microglia were activated with phosphate-buffered saline (PBS, Servicebio, Cat# G4202) or LPS (100 μg/ml; Sigma-Aldrich, Darmstadt, Germany) in the presence or absence of (10, 20, 40) μg/ml GRb1. Some cultures were pretreated for 1 h with 10 µM GW9662, then treated with GRb1 for 24 h, and nally with LPS for 24 h. The collected microglia were transferred to a 6-well plate (2 × 10 5 cells/cm 2 ) for subsequent analysis.
Tissues were enzymatically dissociated into a single-cell suspension using 0.25% pancreatin (Gibco, California, USA), then the mixed glial cells were cultured for 1 week in DMEM/F12 (Gibco, California, USA, Cat# C11330500BT) containing 10% fetal bovine serum (Gibco, California, USA) at 37 °C and 5% CO 2 .
The isolated microglia were activated with phosphate-buffered saline (PBS, Servicebio, Cat# G4202) or LPS (100 μg/ml; Sigma-Aldrich, Darmstadt, Germany) in the presence or absence of (10, 20, 40) μg/ml GRb1. Some cultures were pretreated for 1 h with 10 µM GW9662, then treated with GRb1 for 24 h, and nally with LPS for 24 h. The collected microglia were transferred to a 6-well plate (2 × 10 5 cells/cm 2 ) for subsequent analysis.
3
Isolation and Differentiation of Human Atrial Cardiomyocytes
4
Isolation of Stromal Fibroblasts from FRT Tissue
5
Isolation of Murine Cardiac Fibroblasts
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Wild type C57BL/6 mice used for CFs isolation were purchased from Model Animal Research Center of Nanjing University (Nanjing, China). Mouse cardiac fibroblasts (CFs) were isolated from heart of 8–9 weeks old C57BL/6 mice. Ventricles were minced and digested in 0.05% collagenase and 0.05% pancreatin (Gibco, Invitrogen, Carlsbad, CA, USA) containing PBS solution at 37°C and waved at 220 rpm 10 min for 4–5 times. Cells were pre-plated for 1 hour on uncoated culture dishes (Corning Inc., NY, USA), during which CFs rapidly adhered to the dishes. After preplating the medium containing cardiomyocytes was removed and the attached CFs were washed and further cultured in DMEM (Gibco) containing 10% fetal calf serum (FCS, Gibco) and 10% bovine calf serum (BCS, Gibco) at 37°C and 5% CO2. Fresh complete medium was added and replaced every 3 days. Each primary culture was subcultured 1∶2 when CFs grew to approximately 80%–90% confluence. Fibroblasts up to passage 3 identified by fibroblast specific protein 1 (FSP-1), vimentin, α-SMA, CD31 and Troponin T immunofluorescence staining (sup Fig. 1 ) were used in subsequent studies.
6
A549 Cell Culture and Characterization
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A549 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, Manassas). Dulbecco’s Modified Eagle’s medium (DMEM), fetal calf serum, and pancreatin were purchased from Invitrogen (Invitrogen, Waltham, MA, USA). Bicinchoninic acid (BCA) protein quantification kit was purchased from Pierce (PIERCE, Illinois, USA). MatrigelTM basement membrane matrix, Transwell chamber and Matrigel and FACS Vantage SE flow cytometer were purchased from BD Biosciences (BD Biosciences, NJ, USA). Antibodies for Survivin, PCNA, Caspase3, Caspase9, PI3K, p-PI3K, AKT and p-AKT were purchased from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA). HRP-labeled goat anti-rat secondary antibody was purchased from Santa Cruz (Santa Cruz Biotechnology Dallas, TX, USA). Apoptosis detection kit (Annexin PE/7-AAD) was purchased from Kaiji (Nanjing Kaiji Biological Co., Ltd., China). High speed refrigerated centrifuge was from Beckman (Beckman, Coulter, CA, USA). CO2 incubator was from Thermo (Thermo Fisher Scientific, Waltham, MA, USA). Electrophoresis chamber and Trans-Blot Turbo were purchased from Bio-Rad (Bio-Rad, Hercules, CA, USA). Gel imaging system GDS-800 was purchased from UVP (1UVP, LLC, Upland, CA, USA).
7
Isolation and Differentiation of Fetal Myoblasts
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Early fetal myoblasts were isolated from forelimbs of E16.5 Noggin−/− and control embryos (after careful removal of bones and skin) as previously described40 (link). Briefly, tissues were dissociated one hour at 37 °C with 0.06% Collagenase (from Clostridium Histolyticum, Sigma-Aldrich St. Louis, Mo, USA, for all reagents excepted when notified) and 0.04% Pancreatin in PBS (Gibco-Invitrogen, Carlsbad, CA, USA). After centrifugation, cells were seeded in collagen-coated dishes in presence of Dulbecco’s modified Eagle’s medium (DMEM)-high glucose and 20% fetal bovine serum (FBS; Gibco-Life Technologies, Carlsbad, CA, USA), supplemented with 1% Chicken Embryo Extract (Sera Laboratories International, UK), 100 mg/ml Sodium Pyruvate, 100 IU/ml penicillin and streptomycin, 2 mM L-glutamine. The plates were incubated at 37 °C, 5% CO2, 5% O2 and the medium refreshed every two days. When cells reached confluence, the differentiation was induced by shifting medium to DMEM high glucose supplemented with 2% horse serum (Gibco-Invitrogen, Carlsbad, CA, USA) and 100 mg/ml Sodium Pyruvate (differentiation medium; DM).
8
Isolation and Differentiation of Human Atrial Cardiomyocytes
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Human atrial appendages were obtained from male patients (35 to 90 years old) undergoing cardiac surgery through donation. The protocol received authorization from the University Hospital Ethics Committee and the Cantonal Ethics Committee on research involving humans. Tissues were minced and enzymatically digested in a buffer containing 0.45 mg/ml collagenase (Worthington Biochemical Corporation, Lakewood, New Jersey) and 1 mg/ml pancreatin (Invitrogen, Carlsbad, California) (Supplemental Figure 1A ). Following 24 hours in culture, cells that remained in suspension were discarded, and adherent cells were expanded in expansion medium (3:1 DMEM 1g/l glucose/Medium 199 [Invitrogen] supplemented with 10% horse serum [Serotec, Kidlington, United Kingdom], 5% fetal bovine serum [Serotec], 100 U/ml penicillin [Invitrogen], and 100 μg/ml streptomycin [Invitrogen]). For inducing differentiation, cells were switched to MEM alpha (Invitrogen) containing 2% horse serum, 1 μmol/l dexamethasone (Sigma-Aldrich, St. Louis, Missouri), 50 μg/ml ascorbic acid (Sigma-Aldrich), 10 mmol/l β-glycerophosphate (Sigma-Aldrich), 100 U/ml penicillin (Invitrogen), and 100 μg/ml streptomycin (Invitrogen) (differentiation medium [33] (link)), and cultured for 2 to 3 weeks before analysis.
9
Bioactive Compounds Evaluation in Berries
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Blueberry powder and blackcurrant powder were purchased online (Viberi, Timaru, New Zealand). Oat bran was obtained from the local supermarket (Sun Valley, Christchurch, New Zealand). Pepsin (EC 3.4.23.1) pancreatin (EC 232-468-9), α-amyloglucosidase (EC 3.2.1.3), invertase (EC 3.2.1.26), 2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 2,4,6-Tris(2-pyridyl)-s-triazine (TPTZ), 2-Diphenyl-1-picrylhydrazyl (DPPH), 3,5-Dinitrosalicylic acid (DNS, 98%, ACROS Organics™, Waltham, MA, USA ), Folin and Ciocalteu’s phenol reagent, 2,7 dichlorodihydrofluorescein-diacetate (DCFH-DA), gallic acid, rutin, and trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) were all purchased from Sigma-Aldrich (St. Louis, MO, USA). Other chemicals in this study were of analytical grade.
10
Encapsulation of Probiotic Bifidobacterium Animalis
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The probiotic culture of Bifidobacterium animalis subsp. lactis BB-12 was obtained from Chr. Hansen (Hoersholm, Denmark). Sodium alginate, calcium chloride, xanthan gum, κ-carrageenan, pectin, inulin, glucose, and Tween 80 were provided by Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany); glycerol was purchased from Lach-Ner (Brno, Czech Republic); and nanocrystalline cellulose (CNC) from CelluForce (Montreal, Canada). Olive pomace oil was kindly provided by MINERVA S.A. (Athens, Greece) and sweet whey with the following specifications: humidity 1%, fat content 1%, protein content 10.2%, lactose (hydrated) 75%, ash content 7.3%, was kindly provided by ION S.A. (Piraeus, Greece). Whole-fat milk was obtained from the local market. The materials for the microbiological analyses, such as MRS agar, Ringer’s solution, citric acid, and disodium phosphate, were acquired from Merck (Taufkirchen, Germany), whereas l -cysteine-HCl, neomycine sulfate, nalidixic acid, lithium chloride, and paromomycine sulfate by Thermo Fischer Scientific (MA, USA). Pepsin, pancreatin, and bile extract were obtained from Acros Organics (New Jersey, USA).
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