Collagenase p solution

Manufactured by Roche

Sourced in United Kingdom, Germany, United States

Collagenase P solution is a laboratory reagent used for the enzymatic dissociation of tissues. It contains the enzyme collagenase, which breaks down the collagen components of the extracellular matrix, facilitating the isolation of cells from various tissue types.

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Lab products found in correlation

Histopaque density centrifugation, by Merck Group (4 mentions) Histopaque 1077, by Merck Group (3 mentions) Histopaque 1119, by Merck Group (3 mentions) Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (3 mentions) Fetal bovine serum (fbs), by Euroclone (2 mentions) Histopaque gradient, by Merck Group (2 mentions) Hanks balanced salt solution (hbss), by Merck Group (2 mentions) Bovine serum albumin (bsa), by Merck Group (2 mentions) Enzyme free cell dissociation buffer, by Thermo Fisher Scientific (2 mentions) Complete rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions) Optiprep density gradient medium, by Merck Group (1 mentions) Ficoll, by Cytiva (1 mentions) Soybean trypsin inhibitor, by Merck Group (1 mentions) Dispase 2, by Roche (1 mentions) Cell adherent reagent, by Applygen (1 mentions) Penicillin, by Thermo Fisher Scientific (1 mentions) Streptomycin, by Thermo Fisher Scientific (1 mentions) Rpmi 1640, by Roche (1 mentions) Pyruvate, by Thermo Fisher Scientific (1 mentions) Heat inactivated fcs, by Harvard Bioscience (1 mentions)

Spelling variants of this product

Collagenase P (567 citations) Collagenase (359 citations) Collagenase solution (9 citations)

20 protocols using collagenase p solution

Isolation and Culture of Pancreatic Islets

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Male Lewis rats (Harlan Laboratories, Italy) 12 weeks of age were used as donors of pancreatic islets. Animal care and treatment were conducted in conformity with the institutional guidelines, in compliance with national (D.L. vo n. 26/2014) and international (European Union Directive 2010/63/UE: Guide for the Care and Use of Laboratory Animals, U.S National Research Council, 1996) laws and policies. The protocol was approved by the Ethic Committee of Mario Negri Institute for Pharmacological Research (n° BG01/C). All the experiments were repeated at least three times to validate the results.
Islets were isolated from the pancreas of Lewis rats (body weight 250∼300 g), using an automatic procedure. Briefly, the pancreas of anesthetized rats were distended with collagenase P solution (Boehringer-Mannheim, Mannheim, Germany), removed and then loaded into a digestion chamber at 37℃. When optimum digestion time was reached, the chamber was flushed with 4℃ Hanks’ balanced salt solution (HBSS, Gibson Nitrogen Corporation, Paisley, Scotland) and digested tissue was purified by centrifugation on a Histopaque gradient (1.077 g/ml, Sigma, St. Louis, MO). Islets were cultured at 37℃ in a humidified atmosphere containing 5% CO₂ in RPMI 1640 medium (Life Technologies Italia, Monza, Italy), supplemented with 10% fetal bovine serum (EuroClone, Pero MI, Italy) (15 (link)).

Fumagalli G., Monfrini M., Donzelli E., Rodriguez-Menendez V., Bonandrini B., Figliuzzi M., Remuzzi A., D’Amico G., Cavaletti G, & Scuteri A. (2019). Protective Effect of Human Mesenchymal Stem Cells on the Survival of Pancreatic Islets. International Journal of Stem Cells, 13(1), 116-126.

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Isolation of Pancreatic Islets from Lewis Rats

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Male Lewis rats (Harlan Laboratories, Italy) 12 weeks of age were used as donors of pancreatic islets. Animal care and treatment were conducted in conformity with the institutional guidelines, in compliance with national (DL n. 116/1992, Circ. n. 8/1994) and international (EEC Council Directive 86/609, OJL 358, Dec 1987; NIH Guide for the Care and Use of Laboratory Animals, US NRC, 1996) laws and policies. The protocol was approved by the Ethic Committee of Mario Negri Institute for Pharmacological Research (n° BG01/C). All the experiments were repeated at least three times to validate the results.
Islets were isolated from the pancreas of Lewis rats (body weight 250–300 g), using an automatic procedure. Briefly, the pancreas of anesthetized rats were distended with collagenase P solution (Boehringer -Mannheim, Mannheim, Germany), removed and then loaded into a digestion chamber at 37°C. When optimum digestion time was reached, the chamber was flushed with 4°C Hanks' balanced salt solution (HBSS, Gibson Nitrogen Corporation, Paisley, Scotland) and digested tissue was purified by centrifugation on a Histopaque gradient (1.077 g/mL, Sigma, St. Louis, MO). Islets were cultured at 37°C in an atmosphere of humidified air +5% CO₂ in RPMI 1640 medium (Life Technologies Italia, Monza, Italy), supplemented with 10% fetal bovine serum (EuroClone, Pero MI, Italy).

Scuteri A., Donzelli E., Rodriguez-Menendez V., Ravasi M., Monfrini M., Bonandrini B., Figliuzzi M., Remuzzi A, & Tredici G. (2014). A Double Mechanism for the Mesenchymal Stem Cells' Positive Effect on Pancreatic Islets. PLoS ONE, 9(1), e84309.

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Isolation and Purification of Pancreatic Islets

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Islets were isolated via collagenase P perfusion of the pancreas^{31 (link)}. In brief, collagenase P solution (1 mg ml⁻¹, Roche) was injected through the ampulla of Vater and pancreata were digested at 37.5 °C for 12 min. Digestion was stopped by addition of ice-cold HBSS (Thermo Fisher), including 0.05% (w/v) BSA (Sigma-Aldrich). The tissue suspension was centrifuged and islets were purified by density gradient purification using 15% OptiPrep density gradient medium (Sigma-Aldrich). Islets in the visible density gradient layer were collected, rinsed with HBSS and incubated in complete RPMI-1640 medium (Gibco) at 37 °C with 5 % CO₂.

Liskiewicz A., Khalil A., Liskiewicz D., Novikoff A., Grandl G., Maity-Kumar G., Gutgesell R.M., Bakhti M., Bastidas-Ponce A., Czarnecki O., Makris K., Lickert H., Feuchtinger A., Tost M., Coupland C., Ständer L., Akindehin S., Prakash S., Abrar F., Castelino R.L., He Y., Knerr P.J., Yang B., Hogendorf W.F., Zhang S., Hofmann S.M., Finan B., DiMarchi R.D., Tschöp M.H., Douros J.D, & Müller T.D. (2023). Glucose-dependent insulinotropic polypeptide regulates body weight and food intake via GABAergic neurons in mice. Nature Metabolism, 5(12), 2075-2085.

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Isolation and Characterization of Rat and Human Islets

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Rat islets. All protocols were approved by the Ethical Committee [2020/UCL/MD/016]. Male Wistar rats were put under inhalatory anesthesia and euthanized by exsanguination after laparotomy. 10 ml of Collagenase P solution (1 U/ml – Roche, #11213873001) was injected into the pancreas through the bile duct, under direct observation, to ensure homogeneous distribution throughout the organ. The pancreas was then dissected free and stored in ice-cold Krebs awaiting processing.
Digestion was done by putting the pancreas at 37°C in a water bath for 15 minutes. Digestion was controlled by direct observation and was stopped by adding cold Krebs, followed by 3–4 washes. Islets were then handpicked under a dissection microscope.
Human islets were procured at Tebubio. Two batches were used in our study: 58 years old, BMI 29, HbA1c 5.0%, and 32 years old, BMI 26.9, HbA1c 4.9%. After reception, they were cultured for 48 hours, as instructed by the provider, to allow for recovery.

Ramirez M., Bastien E., Chae H., Gianello P., Gilon P, & Bouzin C. (2024). 3D evaluation of the extracellular matrix of hypoxic pancreatic islets using light sheet fluorescence microscopy. Islets, 16(1), 2298518.

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Isolation of Murine and Human Pancreatic Islets

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Islets from murine pancreata were isolated as described in our earlier publication^{34 (link)}. In brief, a total of 3 mL collagenase P solution (0.4 mg/mL, Cat#11213873001, Roche) was slowly injected into the common bile duct after occlusion of the distal end proximal to the duodenum. The distended pancreas was excised and digested with collagenase P at 37 °C for 15 min. The collagenase digest was then subjected to density gradient centrifugation in Histopaque-1077 (Cat#10771, Sigma) to facilitate the harvest of islets. Islets were finally hand-picked using a micropipette under stereomicroscope. For human islets isolation, pancreas was stored and transported in organ perfusion fluid. After removal of all extraneous connective tissue and fat, pancreas was hand distended for 5 min with 2 mg/ml collagenase P solution supplemented with 20 mg/ml bovine serum albumin (BSA, Cat#A1933, Sigma) and 2 mg/ml soybean trypsin inhibitor (Cat#T9003, Sigma). Following the complete distension of the pancreas with enzyme solution, the organ was chopped into pieces and digested at 37 °C for 20 min. The collagenase digest was then subjected to Ficoll (Cat#17144002, Cytiva) density gradient separation. Islets were finally hand-picked using a micropipette under stereomicroscope.

Liu R., Liu C., He X., Sun P., Zhang B., Yang H., Shi W, & Ruan Q. (2022). MicroRNA-21 promotes pancreatic β cell function through modulating glucose uptake. Nature Communications, 13, 3545.

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Isolation of Pancreatic Islet Cells

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Pancreatic lymph nodes (PLNs) were disrupted mechanically with a 30G needle. Bone-marrow cells were flushed out from the hind legs (femur and tibia). Spleens were homogenised and erythrocytes were lysed. Pancreases were inflated with collagenase P solution (Roche, Burgess Hill, UK) in Hanks’ Balanced Salt Solution (HBSS) via the common bile duct, followed by collagenase digestion with shaking at 37°C for 10 min. Islets were isolated by Histopaque density centrifugation (Sigma-Aldrich, Gillingham, UK), hand-picked under a dissecting microscope and trypsinised to generate single-cell suspensions. Islet cells were rested at 37°C, 5% CO₂ in complete Iscove’s Modified Dulbecco’s Medium (IMDM) overnight, before stimulation for intracellular staining.

Da Rosa L.C., Boldison J., De Leenheer E., Davies J., Wen L, & Wong F.S. (2018). B cell depletion reduces T cell activation in pancreatic islets in a murine autoimmune diabetes model. Diabetologia, 61(6), 1397-1410.

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Isolation and Purification of Pancreatic Islets

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Each pancreas was carefully removed and placed immediately in 10 ml washing solution, which was made up of 5% fetal calf serum and 10 mM Hepes in 0.1 M phosphate buffered saline (PBS). The pancreas was cut into smaller pieces and placed in a 15-ml tube containing 2 ml collagenase P solution (1.4 mg/ml, 1129002001, Roche, Indianapolis, IN, USA) on ice. Once the collections were done, they were pooled and incubated for 10 min at 37°C with intermittent vigorous shaking. Approximately 10 ml G solution (HBSS, 0.35 g NaHCO₃/L and 1% BSA) was then added and the mixture centrifuged for 2 min at 100 g at 4°C. The pellet arising from the centrifugation was re-suspended in 10 ml G solution and then filtered through a size 40 (420 µm) sieve into a 50-ml conical tube. The total volume was made up to 10 ml with G solution and centrifuged at 130 g to reform the pellet. The pellet was then re-suspended in 10 ml histopaque 1100 solution (120 ml histopaque 1077 (10771, Sigma-Aldrich, St Louis, MO, USA) and 120 ml histopaque 1119 (11191, Sigma-Aldrich) and centrifuged for 30 min at 210 g. The supernatant containing islets was transferred to a new 50-ml conical tube and centrifuged for 5 min at 300 g. The pelleted islets were washed in 10 ml G solution, centrifuged at 130 g at 4°C for 10 min and genomic DNA isolated.

Prabakaran A.D., Karakkat J.V., Vijayan R., Chalissery J., Ibrahim M.F., Kaimala S., Adeghate E.A., Al-Marzouqi A.H., Ansari S.A., Mensah-Brown E, & Emerald B.S. (2018). Identification of early indicators of altered metabolism in normal development using a rodent model system. Disease Models & Mechanisms, 11(3), dmm031815.

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Islet Isolation from Murine Pancreas

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The pancreas was perfused with Collagenase P solution (Roche) (35 day old and adult mice: 3 ml; 18 day old mice: 1 ml) through the common bile duct. Pancreata were harvested and digested at 37 °C for 18 min (35 day old and adult mice) or 15 min (18 day old mice). Islets were hand-picked under a stereo microscope and incubated at 37 °C overnight in a humidified atmosphere of air and 5% CO₂ in RPMI-1640 medium (11.1 mM glucose) supplemented with 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin sulfate.

Nicholas L.M., Valtat B., Medina A., Andersson L., Abels M., Mollet I.G., Jain D., Eliasson L., Wierup N., Fex M, & Mulder H. (2017). Mitochondrial transcription factor B2 is essential for mitochondrial and cellular function in pancreatic β-cells. Molecular Metabolism, 6(7), 651-663.

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Isolation and Culture of Rat Islets and Beta Cells

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Pancreatic islets and β cells were obtained from rats that had been anesthetized and then euthanized, as described previously (Gao, et al., 2016 (link)). Briefly, islets were separated by injecting collagenase P solution (1 mg/ml; Roche, Indianapolis, IN, United States), then digested at 37°C and subjected to density gradient centrifugation using Histopaque-1077 (Sigma-Aldrich, St. Louis, MO, United States). Single β cells were obtained from islets by dispase Ⅱ (Roche) digestion and seeded on small slides pretreated with cell adherent reagent (Applygen Technologies Inc., Beijing, China). Isolated rat islets and β cells were cultured in RPMI-1640 (Hyclone, South Logan, UT, United States) medium containing 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, United States), in a humidified atmosphere of 5% CO₂ at 37°C.

Liu Z., Yang H., Zhi L., Xue H., Lu Z., Zhao Y., Cui L., Liu T., Ren S., He P., Liu Y, & Zhang Y. (2021). Sphingosine 1-phosphate Stimulates Insulin Secretion and Improves Cell Survival by Blocking Voltage-dependent K+ Channels in β Cells. Frontiers in Pharmacology, 12, 683674.

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Isolation and Culture of Murine Pancreatic Islets

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RINm5F Insulinoma cells (ATCC, Wesel, Germany) were maintained in RPMI 1640 supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin (Life Technologies, Darmstadt, Germany), and 10% heat-inactivated FCS (Biochrom, Berlin, Germany) at 37°C and 5% CO₂. For experiments, cells were seeded on 6-well and 96-well polystyrene plates (Greiner, Frickenhausen, Germany) in aforementioned culture medium.
Murine islets of Langerhans were isolated from wildtype C57Bl/6J mice and cultured at 37°C and 5% CO₂ in RPMI 1640 supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, 1% non-essential amino acids, 1% pyruvate (Life Technologies), and 10% heat-inactivated FCS (Biochrom). Briefly, the pancreas was dissected and perfused with collagenase P solution (1.2 U/ml in RPMI 1640, Roche Applied Science, Mannheim, Germany). Islets were isolated by density gradient centrifugation using Ficoll Paque Plus (GE Healthcare, München, Germany), were washed, and handpicked thereafter. Isolation by combined density gradient centrifugation and subsequent handpicking as performed herein results in highly pure cultures with islet purity >99% (Ramírez-Domínguez and Castaño, 2015 (link)). After 48–96 h recovery in aforementioned medium, 60–100 islets (as specified in the respective figure legends) were seeded on 12-well polystyrene plates (Greiner, Frickenhausen).

Berner A., Bachmann M., Bender C., Pfeilschifter J., Christen U, & Mühl H. (2016). Though Active on RINm5F Insulinoma Cells and Cultured Pancreatic Islets, Recombinant IL-22 Fails to Modulate Cytotoxicity and Disease in a Protocol of Streptozotocin-Induced Experimental Diabetes. Frontiers in Pharmacology, 6, 317.

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