Collagenase type 8

Manufactured by Merck Group

Sourced in United States, United Kingdom

Collagenase type VIII is a purified enzyme derived from Clostridium histolyticum. It is designed for use in cell isolation and tissue dissociation applications.

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Lab products found in correlation

96 protocols using collagenase type 8

Isolation of Colonic Lamina Propria Cells

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Colonic lamina propria (cLP) cells were isolated as previously described^{16 (link)}. Briefly, colons were excised, opened longitudinally, washed of fecal contents and then cut into 1 cm pieces. Intestinal pieces were transferred into Hank’s Balanced Salt Solution (HBSS) medium (Thermo Fisher Scientific), supplemented with 2 mM EDTA, and were shaken for 8 min at 37°C. The remaining tissue was washed, minced and subsequently incubated in digestion medium consisting of RPMI 1640 (Thermo Fisher Scientific), 5% FBS, 0.5 mg/ml collagenase type VIII (Sigma-Aldrich), 5 U/ml DNase (Sigma-Aldrich), 100 IU/ml penicillin and 100 μg/ml streptomycin (Thermo Fisher Scientific), for 40 min at 37°C by gentle shaking at a speed of 150 rpm. The cell suspensions were filtered through a 100 μm mesh and centrifuged at 1700 rpm. The obtained cells were filtered through a 70 μm filter, washed twice with PBS and used as cLP cells.

Li X.V., Leonardi I., Putzel G.G., Semon A., Fiers W.D., Kusakabe T., Lin W.Y., Gao I.H., Doron I., Gutierrez-Guerrero A., DeCelie M.B., Carriche G.M., Mesko M., Yang C., Naglik J.R., Hube B., Scherl E.J, & Iliev I.D. (2022). Immune regulation by fungal strain diversity in Inflammatory Bowel Disease. Nature, 603(7902), 672-678.

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Isolation of Thymocytes and Intestinal T Cells

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To obtain thymocytes or T cells, thymi or peripheral lymphoid organs (spleen, peripheral/mesenteric lymph nodes) were dissociated through a 40-μm nylon mesh into RPMI (supplemented with 10% FBS). Cells were washed with FACS buffer (2% FBS, 2% NaN₃, 2 mM EDTA) prior to antibody staining. For analysis of T cells from the lamina propria (LP), the large intestine (extending from the rectum to the cecum) was filleted, the feces removed and the colon washed in PBS by vigorous shaking for 2 minutes. The tissue was incubated in 1 mM DTT/PBS with gentle agitation for 10 minutes at room temperature, followed by two incubations in 30 mM HEPES/10 mM EDTA PBS solution at 37°C for 10 minutes each, shaking at 200 rpm. The tissue was then transferred to complete medium (RPMI + 10% FBS) supplemented with collagenase type VIII (200 CDU per ml; Sigma Aldrich) and DNase I (150 ug/ml; Sigma Aldrich) and incubated for 90 minutes at 37 °C. The tissue was further dissociated by vigorously shaking and filtering through 100μm nylon mesh. To isolate T cells from the homogenate, the digested tissue was resuspended in 40% Percoll solution, overlaid onto 80% Percoll solution and centrifuged at 600 x g for 20 minutes at room temperature. Single cell suspensions that included T cells were isolated by collecting cells at the interface between the two Percoll solutions.

O’Hagan K.L., Choi J., Pryshchep O., Chernoff J, & Phee H. (2015). Pak2 links TCR signaling strength to the development of regulatory T cells and maintains peripheral tolerance. Journal of immunology (Baltimore, Md. : 1950), 195(4), 1564-1577.

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Isolation of Intestinal Lamina Propria Macrophages from Septic Mice

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Lamina propria macrophages from the intestinal mucosa of septic mice were isolated as previously reported^{49 (link), 50 (link)}. Briefly, the tissues were first predigested to eliminate epithelial cells. The intestinal tissues were then digested with digestion solution (1 mg/mL collagenase type VIII + 3 mg/mL dispase II + 40 μg/mL DNase I + 5% FBS) + Hanks Solution (all reagents were from Sigma-Aldrich, St Louis, MO, USA). After incubation at 37 °C for 30 min while shaking (150 rpm), the tissues were filtered through a 100-μm nylon mesh filter (Fisher brand, Loughborough, UK). The remaining pieces were repeatedly digested and filtered, and the filtered suspensions were rinsed with PBS. The cell suspensions were centrifuged, resuspended in fresh PBS, and filtered again through a 40-μm nylon mesh filter (Fisher brand, Loughborough, UK).

Wu X., Ren J., Chen G., Wu L., Song X., Li G., Deng Y., Wang G., Gu G, & Li J. (2017). Systemic blockade of P2X7 receptor protects against sepsis-induced intestinal barrier disruption. Scientific Reports, 7, 4364.

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Inflammatory Pathway Activation Assay

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LPS purified from Escherichia coli O111:B4, TNBS, t-BHP, collagenase type VIII, RPMI 1640 were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Antibodies for COX-2, ERK, p-ERK, IκBα, p- IκBα, IRAK1, p-IRAK1, iNOS, p65, p-p65, TAK1 and p-TAK1 were purchased from Cell Signaling Technology (Beverly, MA, U.S.A.). Radioimmuno-precipitation assay (RIPA) buffer, tetramethyl benzidine was purchased from Sigma (St Louis, MO, U.S.A.). ELISA kits for IL-1β, IL-6, IL-10, and TNF-α were purchased from R&D Systems (Minneapolis, MN, U.S.A.). mRNA isolation kit was purchased from Qiagen (Hilden, Germany). A diazo-coupled limulus amoebocyte lysate (LAL) assay kit was purchased from Cape Cod Inc. (E. Falmouth, MA, USA). Pan T Cell Isolation Kit II was purchased from MiltenyiBiotec GmbH (Bergisch Gladbach, Germany). Anti-CD28, anti-CD3, recombinant IL-6, and recombinant TGF-β were purchased from BioGems International Inc. (Westlake Village, CA, U.S.A.). Fetal bovine serum (FBS) and heat-inactivated fetal calf serum (FCS) purchased from Panbiotech GmbH (Aidenbach, Germany). de Man, Rogosa and Sharpe (MRS) medium for probiotics was purchased from BD (Sparks, MD, USA). General anaerobic medium (GAM) for probiotics and other bacteria were purchased from Nissui Pharmaceutical Co (Tokyo, Japan). Other chemicals used were of the highest grade available.

Jang S.E., Jeong J.J., Kim J.K., Han M.J, & Kim D.H. (2018). Simultaneous Amelioratation of Colitis and Liver Injury in Mice by Bifidobacterium longum LC67 and Lactobacillus plantarum LC27. Scientific Reports, 8, 7500.

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Isolation and Analysis of Th17 and Treg Cells

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For the flow cytometric analysis of Th cells in the lamina propria, colons were squashed and sequentially incubated with 2.5 mM EDTA at 37°C with shaking for 20 min, and 1 mg/mL collagenase type VIII (Sigma, St. Louis MO, USA) for 20 min at 37°C (Lee et al., 2015b (link)). The reaction mixture were filtered and T cells were isolated using a Pan T cell Isolation Kit II, fixed, stained with anti-Foxp3 or anti-IL-17A antibodies (Biogems, CA, USA) and analyzed by flow cytometry.
For the flow cytometric analysis of Th17 and Treg cells in the spleens, spleens were crushed into a single-cell suspension, lysed with Tris-buffered ammonium chloride, suspended in RPMI 1640 medium, and then filtered, and the T cells were isolated using a Pan T cell Isolation Kit II (Lee et al., 2015a (link)). Isolated T cells were fixed and stained with anti-FoxP3 or anti-IL-17A antibodies and then analyzed by flow cytometry.

Lim S.M., Lee S.Y., Jeong J.J., Choi H.S., Chang H.B, & Kim D.H. (2016). DW2007 Ameliorates Colitis and Rheumatoid Arthritis in Mice by Correcting Th17/Treg Imbalance and Inhibiting NF-κB Activation. Biomolecules & Therapeutics, 24(6), 638-649.

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Phenotyping and Cell Cycle Analysis

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Rabbit anti-rat CD29-FITC, CD34-FITC, CD44-FITC, and CD45-FITC were used to identify the BM-MSCs phenotypes. All antibodies were purchased from BD Biosciences (Franklin Lakes, NJ, USA).
For cell cycle detection, IEC-6 cells were washed twice with PBS and fixed in cold ethanol for 30min, and then incubated with propidium iodide (PI) for 30min. Thereafter, cells (1–2 × 10⁵) were analyzed by flow cytometry (BD Bioscience). Cell death was measured using the AnnexinV-FITC/PI Apoptosis Detection Kit (BD Bioscience).
For intracellular staining, mesenteric lymph nodes were dissected into small pieces and incubated in RPMI 1640 medium containing collagenase type VIII at 200 U/ml (Sigma-Aldrich) for 50min at 37°C while stirring. Supernatants containing cells were collected and the cells were washed and resuspended in complete RPMI 1640. Cells were incubated with phorbol myristate acetate (50 ng/ml) and ionomycin (750 ng/ml) for 4 h. Labeled cells were then analyzed on FACS. Monoclonal antibodies against CD4 and Foxp3 were obtained from eBiosciences, San Diego, CA, USA.

Chen H., Min X.H., Wang Q.Y., Leung F.W., Shi L., Zhou Y., Yu T., Wang C.M., An G., Sha W.H, & Chen Q.K. (2015). Pre-activation of mesenchymal stem cells with TNF-α, IL-1β and nitric oxide enhances its paracrine effects on radiation-induced intestinal injury. Scientific Reports, 5, 8718.

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Isolation and Culture of Rheumatoid Arthritis Synovial Fibroblasts

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This study was approved by the Institutional Review Board of University of California, San Diego School of Medicine and informed consent was obtained from all participants. Synovial tissue was obtained from patients with RA at the time of total joint replacement or synovectomy as previously described. The diagnosis of RA conformed to American College of Rheumatology 1987 revised criteria. The samples were processed for cell culture. The synovium was minced and incubated with 1 mg/ml collagenase type VIII (Sigma) in serum-free RPMI 1640 (Gibco BRL, Grand Island, NY, USA) for 1 h at 37°C, filtered, extensively washed, and cultured in DMEM (Gibco BRL) supplemented with 10% FBS (Gemini Bio Products, Calabasas, CA, USA), penicillin, streptomycin, gentamicin, and glutamine, in a humidified 5% CO₂ atmosphere. Cells were allowed to adhere overnight, non-adherent cells were removed, and adherent FLS were split at 1:3 when 70 to 80% confluent. FLS were used from passage 3 through 9 during which time they are a homogeneous population of cells (<1% CD11b-positive, <1% phagocytic, and <1% FcγRII- and FcγRIII-receptor-positive). FLS were cultured and used at 80% confluence. Cells were synchronized in 0.1% FBS for 24 h before the addition of the appropriate stimulus.

Gong W., Kolker S.J., Usachev Y., Walder R.Y., Boyle D.L., Firestein G.S, & Sluka K.A. (2014). Acid-sensing ion channel 3 decreases phosphorylation of extracellular signal-regulated kinases and induces synoviocyte cell death by increasing intracellular calcium. Arthritis Research & Therapy, 16(3), R121.

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Extraction and Analysis of Adipose Tissue

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Adipose tissue was dissected, washed in PBS, minced, and digested for 50 min at 37 °C in 0.1% (w/v) collagenase type VIII (Sigma–Aldrich, St. Louis, MO) with gentle shaking. Tissue suspensions were passed through a 100-μm cell strainer and centrifuged at 500 g for 5 min to pellet the SVF. The floating adipocytes were transferred to a fresh tube and the pellet containing SVFs was resuspended with ammonium chloride lysis buffer to remove red blood cells. Lysis buffer containing 50 mM Tris–HCl (pH 6.8), 2% SDS, phosphatase inhibitors (10 mM Na₃VO₄ and 10 mM NaF), and a protease inhibitor mixture (Roche Applied Science) was added to the pellet containing SVF or transferred adipocyte fraction for protein extraction. Equal amounts of protein were subjected to SDS-PAGE and immunoblotted using specific primary antibodies.

Xing C., Jiang D., Liu Y., Tang Q, & Huang H. (2020). Lysyl oxidase inhibition enhances browning of white adipose tissue and adaptive thermogenesis. Genes & Diseases, 9(1), 140-150.

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Isolation and Characterization of Lung Immune Cells

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A portion of the perfused right lung lobes was mechanically digested and incubated for 30 min at 37°C at 5% CO₂ in RPMI 1640 medium (Gibco) supplemented with glutamine, Na-pyruvate, 2-ME, penicillin, streptomycin, 10% heat-inactivated FCS, collagenase D (Roche), and collagenase type VIII (Sigma-Aldrich). Single-cell suspensions of lungs were then prepared by meshing the lungs through 40-μm nylon cell strainers and red blood cell lysis. Viable cells were counted by trypan blue exclusion. Cells were blocked with anti-mouse CD16/CD32 mAb (BioLegend) and human Fc receptor blocking solution (BioLegend), and then surface stained with monoclonal antibodies purchased from BioLegend: anti-mouse CD45 (clone 30-F11) anti-human CD45 (clone HI30), anti-human CD3 (clone UCHT1), anti-human CD4 (clone OKT4), anti-human CD8 (clone SK-1), anti-human CD19 (clone HIB19), anti-human CD20 (clone 2H7), anti-human CD33 (clone WM53), anti-human CD66a/c/e/b (clone ASL-32 and G10F5), anti-human CD14 (clone M5E2), and anti-human CD11c (clone Bu15) for 30 min at 4°C. The samples were analyzed by flow cytometry using a BD FACS CANTO II in the biosafety level 3 and visualized using FlowJo software.

Arrey F., Löwe D., Kuhlmann S., Kaiser P., Moura-Alves P., Krishnamoorthy G., Lozza L., Maertzdorf J., Skrahina T., Skrahina A., Gengenbacher M., Nouailles G, & Kaufmann S.H. (2019). Humanized Mouse Model Mimicking Pathology of Human Tuberculosis for in vivo Evaluation of Drug Regimens. Frontiers in Immunology, 10, 89.

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Intestinal Lamina Propria Cell Isolation

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Intestinal lamina propria cells were isolated from colonic resection specimens from control and DSS mice. Briefly, intestinal resections were washed and then cut into 1 mm² pieces, then incubated in a buffer consisting of RPMI, 75 mg/mL collagenase type VIII (Sigma), DNase (Sigma), and HEPES for 45 minutes in a C shaker. We collected the supernatant by centrifugation at 13,000g at 4°C.

Khan S., Raj D., Sahu S., Naseer A., Singh N.C., Kumari S., Ishteyaque S., Sharma J., Lakra P., Mugale M.N., Trivedi A.K., Srivastava M., Chandra T., Bhosale V., Barthwal M.K., Gupta S.K., Mitra K., Nazir A., Ghoshal U.C, & Lahiri A. (2023). CLUH functions as a negative regulator of inflammation in human macrophages and determines ulcerative colitis pathogenesis. JCI Insight, 8(11), e161096.

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