Animals were euthanized, and the abdomen was subsequently incised. The pancreas was exposed and infused through the main bile duct with collagenase type V (SigmaAldrich, St. Louis, MO) suspended at 0.7 mg/mL in a buffer solution (Perfusion Solution, Cellgro-Mediatech, Herndon, VA). The distended pancreas was removed and digested at 37°C for 12 minutes, followed by vigorous agitation for 10 seconds. After washing, islet purification was obtained through centrifugation on a Euro-Ficoll (Corning, Manassas, VA) gradient. Islets were hand-picked under an inverted microscope, plated in supplemented cRPMI medium 1640 [10% fetal bovine serum (FBS; Gemini BioProducts, West Sacramento, CA, USA), 2 mM L-glutamine (Sigma-Aldrich), 1% penicillin–streptomycin (Invitrogen, Carlsbad, CA) in RPMI 1640 (Lonza, Walkersville, MD, USA)], and incubated for 24 h at 37°C in 5% CO2 for recovery.
Collagenase type 5
Manufactured by Merck Group
Sourced in United States, United Kingdom
Collagenase type V is a laboratory reagent used for the enzymatic digestion of collagen. It is a mixture of proteolytic enzymes derived from Clostridium histolyticum that function to break down the collagen structure. The core function of Collagenase type V is to facilitate the isolation and extraction of cells and tissues from collagen-rich environments.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (8 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (6 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (6 mentions)
Trypsin edta, by Thermo Fisher Scientific (5 mentions)
Rpmi 1640, by Thermo Fisher Scientific (5 mentions)
L glutamine, by Merck Group (4 mentions)
Fetal bovine serum (fbs), by Lonza (4 mentions)
Histopaque, by Merck Group (4 mentions)
Dnase 1, by Roche (4 mentions)
Dnase 1, by Merck Group (4 mentions)
Penicillin streptomycin, by Merck Group (3 mentions)
Dnase 1, by Qiagen (3 mentions)
Hyaluronidase, by Merck Group (3 mentions)
Propidium iodide, by Thermo Fisher Scientific (3 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (3 mentions)
Histopaque 1077, by Merck Group (2 mentions)
Nicotinamide, by Merck Group (2 mentions)
Actinomycin d, by Merck Group (2 mentions)
Dnase, by Roche (2 mentions)
Glucose, by Thermo Fisher Scientific (2 mentions)
91 protocols using collagenase type 5
1
Pancreatic Islet Isolation Protocol
2
Establishment of PDAC Patient-Derived Xenografts
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Consent's forms of informed patients were collected and registered in a central database. The tumor tissues used for xenograft development was deemed excess to that required for the patient's diagnosis. Two types of samples were obtained, namely Endoscopic Ultrasound-Guided Fine-Needle Aspiration (EUS-FNA) biopsies from patients with unresecable tumors, and tumor tissues from patients undergoing surgery. PDAC samples were mixed with 100 μl of Matrigel (BD Biosciences) and implanted with a trocar (10 Gauge, Innovative Research of America, Sarasota, FL) in the subcutaneous right upper flank of an anesthetized and disinfected mouse. When tumors reached 1 cm3, mice were sacrificed and tumors were removed.
To obtain primary cell cultures of these tumors, xenografts were splited into several small pieces and processed in a biosafety chamber: after a fine mincing, they were treated with collagenase type V (ref C9263; Sigma) and trypsin/EDTA (ref 25200-056; Gibco, Life Technologies) and suspended in DMEM supplemented with 1% w/w Penicillin/Streptomycin (Gibco, Life Technologies) and 10% Fetal Bovine Serum (Lonza). After centrifugation, cells were re-suspended in Serum Free Ductal Media (SFDM) adapted from Schreiber et al. [25 (link)] and incubated at 37°C in a 5% CO2 incubator.
To obtain primary cell cultures of these tumors, xenografts were splited into several small pieces and processed in a biosafety chamber: after a fine mincing, they were treated with collagenase type V (ref C9263; Sigma) and trypsin/EDTA (ref 25200-056; Gibco, Life Technologies) and suspended in DMEM supplemented with 1% w/w Penicillin/Streptomycin (Gibco, Life Technologies) and 10% Fetal Bovine Serum (Lonza). After centrifugation, cells were re-suspended in Serum Free Ductal Media (SFDM) adapted from Schreiber et al. [25 (link)] and incubated at 37°C in a 5% CO2 incubator.
3
Synthetic Cannabinoid Receptor Assay
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BAR-1 was synthetized by Navarrete-Vázquez and colleagues20 , and its synthesis has been described previously. Dulbecco’s modified Eagle’s medium (DMEM) low glucose, Fetal bovine serum (FBS), penicillin/streptomycin, l -glutamine, collagenase type V, Histopaque 1077, STZ and PCR primers for CB1r, CB2r, MAGL, NAPE-PLD, FAAH, DAGL and 18s rRNA were purchased from Sigma Aldrich (St. Louis MO, USA). Preproinsulin (PPI) and preproglucagon (PPG) were obtained from Qiagen (West Sussex, UK). Real-time PCR master mix and reagents were purchased from Fermentas/Thermo Scientific (Madison, WI, USA) and Invitrogen (Grand Island, NY, USA). A1cNow kit was from ChekDiagnostics (Diagnodistributions, USA), and insulin and glucagon enzyme-linked immunosorbent assay (ELISA) kits were obtained from Alpco (Salem, NH, USA). Anti-insulin primary antibody and secondary antibodies were obtained from Santa Cruz Biotechnology.
4
Isolation and Culture of Porcine Islets
5
Mouse Islet Isolation and Purification
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Mouse islets were isolated using a collagenase-based digestion (Collagenase type V, Sigma-Aldrich, St. Louis, MO), as previously published (35 (link)). Briefly, immediately after euthanasia, 2–3 ml cold collagenase solution (1.95 mg/ml in HBSS) was injected into the pancreas through the common bile duct. The fully inflated pancreas was excised and incubated for 20 min at 37°C in a tissue culture flask, then shaken for 5 s to break up the tissue. The digested tissue was washed 4X with cold HBSS supplemented with 0.2% BSA (HBSS/BSA), and the islets purified on a Ficoll (Type 400) gradient (Amersham, Little Chalfont, UK). Following gradient separation and two washes in HBSS/BSA, the islets were hand-picked and plated in culture dishes in CMRL 1066 medium (Mediatech, Manassas, VA) supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen, Carlsbad, CA), 100 units/ml penicillin, 0.1 mg/ml streptomycin, and 2 mmol/l L-glutamine (Life Technologies, Grand Island, NY). All islets were used between 24 and 48 h after isolation. Each islet batch was composed of a pool of 8–10 mouse donors. Islet viability was determined to be >90% in each batch.
6
Cell Isolation and Microscopy Protocols
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The main reagents for cell isolation and microscopy include: Fluo‐4 AM, propidium iodide and Hoechst‐33342 (ThermoFisher Scientific, Paisley, UK); collagenase type V, inorganic salts and bile salts (all from Sigma‐Aldrich, Gillingham, UK): sodium cholate (NaChol), sodium taurocholate (TC) and taurolithocholic acid 3‐sulfate (TLC‐S). NaHepes buffer was prepared as follows (mm ): NaCl 140, KCl 4.7, Hepes 10, MgCl2 1, glucose 10; pH 7.3. NMDG‐Hepes was a modification of NaHepes, where NaCl was replaced by 140 mm N‐methyl‐d ‐glucamine (NMDG+), pH 7.3.
7
Isolation of Murine Pancreatic Islets
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Islets of Langerhans were isolated from donor C3H mice by collagenase digestion as follows. Mice were euthanized by CO2/O2 suffocation and 2 ml of ice cold RPMI 1640 containing 1 mg/ml collagenase type V (Sigma Aldrich) were infused via the pancreatic duct in situ. After dissection, the perfused pancreata were collected in serum free medium containing collagenase and kept on ice until digestion. Pancreata were digested for 12 min at 37 °C. Digestion was stopped by adding RPMI medium containing 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin and 100 U/ml streptomycin (complete RPMI). After washing, digested pancreata were passed through a mesh. Afterwards, islets were purified on a discontinuous Ficoll gradient (densities: 1.108; 1.096; 1.037; Cellgro by Mediatech Inc, Manassas, VA, USA) by centrifugation at 625 × g for 16 min without brake. Islets were collected from the intersection of the second and third layer and remaining Ficoll was removed by washing with complete RPMI. Isolated islets were cultured overnight in complete RPMI medium in a humidified atmosphere containing 5% CO2 at 37 °C.
8
Endothelial Cell Isolation from Tumor Tissue
9
Pancreatic Islet Isolation and Insulin Secretion Assay
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Pancreatic islets from all groups were isolated by collagenase type V (0.8 mg/mL, Sigma–Aldrich, USA) digestion, as previously described (Bordin et al., 1995 (link)). For static insulin secretion assays, at least four groups of four islets per animal were pre-incubated in 24-wells plates for 45 min at 37°C in 1 mL Krebs-Hepes buffer (115 mM NaCl, 5 mM KCl, 2.6 mM CaCl2, 1 mM MgCl2, 10 mM NaHCO3, 15 mM HEPES, supplemented with 5.6 mM glucose, 3 g/L BSA) under controlled environment (95% O2 + 5% CO2). Afterward, incubation medium was replaced with fresh buffer containing eather 5.6 or 16.7 mM glucose and incubated for one additional hour. In another set of experiments, pancreatic islets from 90-days-old lean not-treated rats were incubated in eather 5.6 or 16.7 mM glucose Krebs-Hepes buffer containing HESc in the concentrations of 10, 30, 100, 300, and 1000 μg/mL for 1 h under same conditions. At the end, plates were cooled on ice bath and supernatants collected and appropriately stored at -20°C for posterior measurement of insulin concentrations by RIA, as earlier described.
10
Isolation of Pancreatic Islets from SD Rats
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Pancreatic islets were isolated from the pancreata of 10–12 week aged-SD rats by the collagenase digestion technique. The Animal Care and Use Committee of DGIST approved all animal protocols. Briefly, the animals were anesthetized and sacrificed by cervical dislocation. Collagenase type V (Sigma, USA) was dissolved to a concentration of 1 mg/ml in Hank's balanced salts solution and transfused into the pancreatic ducts via the common bile duct. The dissected pancreas was then incubated for 10–15 min at 37 °C in a water bath with shaking every 3 min. The islets were isolated under a stereoscope (15×magnification) and placed in Krebs-Ringer bicarbonate buffer (KRBB) containing 0.1% BSA, penicillin (100units/ml), and streptomycin (0.1 mg/ml). The isolated islets were cultured overnight in RPMI-1640 containing 10% FBS and 11.2 mmol/l glucose to ensure optimal recovery and were used for each condition.
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