type iv collagenase  (Millipore)

 
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    Name:
    Collagenase D
    Description:
    Collagenase D is prepared from Clostridium histolyticum cultures by filtration ammonium sulfate precipitation dialysis and lyophilization
    Catalog Number:
    COLLD-RO
    Price:
    None
    Applications:
    Collagenase from C. histolyticum is widely used for the disaggregation of many types of tissues (e.g., lung, heart, muscle, bone, adipose tissue, liver, kidney, cartilage, mammary gland, placentae, blood vessels, brain, tumors) and for the preparation of single cell suspensions for the establishment of primary cell culture systems. Collagenase D is recommended when functionality and integrity of cell-surface proteins are important.Clostridium collagenase from Roche has been used to prepare cells from many types of tissue, such as hepatocytes, adipocytes, pancreatic islets, epithelial cells, muscle cells, endothelial cells, etc. However, suitability of each lot of the enzyme for disruption of a particular tissue should be determined empirically.
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    Structured Review

    Millipore type iv collagenase
    Collagenase D
    Collagenase D is prepared from Clostridium histolyticum cultures by filtration ammonium sulfate precipitation dialysis and lyophilization
    https://www.bioz.com/result/type iv collagenase/product/Millipore
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    type iv collagenase - by Bioz Stars, 2021-04
    97/100 stars

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    Related Articles

    Isolation:

    Article Title: Cooperative function of Pdx1 and Oc1 in multipotent pancreatic progenitors impacts postnatal islet maturation and adaptability
    Article Snippet: .. Isolation of islets from P14 pancreas required collagenase digestion of whole dissected pancreas at 37°C and hand-picking of islets away from exocrine tissue following a Histopaque-1077 (Sigma-Aldrich) gradient. ..

    Injection:

    Article Title: Combination of Alphavirus replicon particle-based vaccination with immunomodulatory antibodies: therapeutic activity in the B16 melanoma mouse model and immune correlates
    Article Snippet: .. Sixteen days after injection, matrigel plugs were resected, incubated for 1 hour at 37°C with 1 mg/ml Collagenase D (Sigma) and dissociated to obtain a single-cell suspension. .. Cells were then stained with the following anti-mouse mAbs: anti-CD45.2-APCCY7, anti-CD3-FITC, anti-NK1.1-APC, anti-CD8-PE–Texas Red, anti-CD4-PE, anti-Foxp3-APC, anti-PD-1 APC, anti-OX40-PE, anti-GITR-PECY7, anti-CD27-PE, anti-ICOS-PE (BD Biosciences) and LIVE/DEAD Fixable Aqua Dead Cell Stain kit (ViD, eBiosciences) or DAPI before acquisition.

    Incubation:

    Article Title: Combination of Alphavirus replicon particle-based vaccination with immunomodulatory antibodies: therapeutic activity in the B16 melanoma mouse model and immune correlates
    Article Snippet: .. Sixteen days after injection, matrigel plugs were resected, incubated for 1 hour at 37°C with 1 mg/ml Collagenase D (Sigma) and dissociated to obtain a single-cell suspension. .. Cells were then stained with the following anti-mouse mAbs: anti-CD45.2-APCCY7, anti-CD3-FITC, anti-NK1.1-APC, anti-CD8-PE–Texas Red, anti-CD4-PE, anti-Foxp3-APC, anti-PD-1 APC, anti-OX40-PE, anti-GITR-PECY7, anti-CD27-PE, anti-ICOS-PE (BD Biosciences) and LIVE/DEAD Fixable Aqua Dead Cell Stain kit (ViD, eBiosciences) or DAPI before acquisition.

    Article Title: Polarization of macrophages in the tumor microenvironment is influenced by EGFR signaling within colon cancer cells
    Article Snippet: Flow cytometry Isolation of primary mouse macrophages from colon was performed as described previously [ ]. .. Briefly, colon tissues were cut into small pieces (1-2 mm) and incubated in 10 mL PRMI 1640 medium with 10 mM HEPES and 5% fetal bovine serum containing 10 mg (1 mg/mL) collagenase D (Sigma-Aldrich), 10 mg (1 mg/mL) dispase II (Roche, Germany), and 100 μL 10 mg/ml DNase I (100 μg/mL) (Sigma-Aldrich) for 30-45 min in a shaking incubator at 37°C. .. For Ana-1 and BMDM cells, single-cell suspensions from mouse colon tissues were incubated with APC-anti-mouse CD11b and FITC-anti-mouse F4/80 (Affymetrix eBioscience, USA), or APC-anti-mouse F4/80 and FITC-anti-mouse CD206 (MMR) (Biolegend, San Diego, CA) antibodies.

    Article Title: Resolution of a chronic viral infection after interleukin-10 receptor blockade
    Article Snippet: .. Splenocytes from mice infected 7 d earlier with LCMV Armstrong or LCMV clone 13 with or without anti–IL-10R antibody treatment were incubated with HBSS medium containing 0.5 mg/ml collagenase D (Sigma-Aldrich) at 37°C, 5% CO2 for 30 min. 0.01 M EDTA was added to disrupt T cell–DC complexes. .. Next, cells were depleted of CD3-expressing cells (Dynal CD3-beads; Dynal), incubated with CD11c microbeads (Miltenyi Biotec), and positively selected using MACS columns.

    Flow Cytometry:

    Article Title: Dysregulated Cytokine Expression by CD4+ T cells from Post-Septic Mice Modulates both Th1 and Th2-Mediated Granulomatous Lung Inflammation
    Article Snippet: .. Flow cytometry Collagenase-digested (Sigma-Aldrich, St. Louis, MO) lung lobes and undigested lymph nodes from animals were processed into single cell suspensions by processing tissues through sterile 40-mm filters, and ammonium chloride lysis buffer was used to eliminate erythrocytes. .. Cells were stained with the following fluorescent antibodies in flow cytometry buffer (phosphate buffered saline, 1% w/v bovine serum albumin, 0.05% w/v sodium azide): Purified αCD16/32 (Fc Block) (2.4G2, BD Biosciences, San Jose, CA), FITC-αCD8a (53–6.7, BD Biosciences), FITC-αCD3ε (145-2C11, BD Biosciences), PeCy7-αCD45 (Ly5, eBioscience, San Diego, CA), PeCy7-αCD3ε (17A2, BioLegend, San Diego, CA), Pacific Blue- (RM4-5, BioLegend) or Pacific Orange (RM4-5, Invitrogen, Carlsbad, CA) -αCD4, and Pacific Blue-αCD45 (30-F11, BioLegend).

    Cytometry:

    Article Title: Dysregulated Cytokine Expression by CD4+ T cells from Post-Septic Mice Modulates both Th1 and Th2-Mediated Granulomatous Lung Inflammation
    Article Snippet: .. Flow cytometry Collagenase-digested (Sigma-Aldrich, St. Louis, MO) lung lobes and undigested lymph nodes from animals were processed into single cell suspensions by processing tissues through sterile 40-mm filters, and ammonium chloride lysis buffer was used to eliminate erythrocytes. .. Cells were stained with the following fluorescent antibodies in flow cytometry buffer (phosphate buffered saline, 1% w/v bovine serum albumin, 0.05% w/v sodium azide): Purified αCD16/32 (Fc Block) (2.4G2, BD Biosciences, San Jose, CA), FITC-αCD8a (53–6.7, BD Biosciences), FITC-αCD3ε (145-2C11, BD Biosciences), PeCy7-αCD45 (Ly5, eBioscience, San Diego, CA), PeCy7-αCD3ε (17A2, BioLegend, San Diego, CA), Pacific Blue- (RM4-5, BioLegend) or Pacific Orange (RM4-5, Invitrogen, Carlsbad, CA) -αCD4, and Pacific Blue-αCD45 (30-F11, BioLegend).

    Lysis:

    Article Title: Dysregulated Cytokine Expression by CD4+ T cells from Post-Septic Mice Modulates both Th1 and Th2-Mediated Granulomatous Lung Inflammation
    Article Snippet: .. Flow cytometry Collagenase-digested (Sigma-Aldrich, St. Louis, MO) lung lobes and undigested lymph nodes from animals were processed into single cell suspensions by processing tissues through sterile 40-mm filters, and ammonium chloride lysis buffer was used to eliminate erythrocytes. .. Cells were stained with the following fluorescent antibodies in flow cytometry buffer (phosphate buffered saline, 1% w/v bovine serum albumin, 0.05% w/v sodium azide): Purified αCD16/32 (Fc Block) (2.4G2, BD Biosciences, San Jose, CA), FITC-αCD8a (53–6.7, BD Biosciences), FITC-αCD3ε (145-2C11, BD Biosciences), PeCy7-αCD45 (Ly5, eBioscience, San Diego, CA), PeCy7-αCD3ε (17A2, BioLegend, San Diego, CA), Pacific Blue- (RM4-5, BioLegend) or Pacific Orange (RM4-5, Invitrogen, Carlsbad, CA) -αCD4, and Pacific Blue-αCD45 (30-F11, BioLegend).

    Mouse Assay:

    Article Title: Resolution of a chronic viral infection after interleukin-10 receptor blockade
    Article Snippet: .. Splenocytes from mice infected 7 d earlier with LCMV Armstrong or LCMV clone 13 with or without anti–IL-10R antibody treatment were incubated with HBSS medium containing 0.5 mg/ml collagenase D (Sigma-Aldrich) at 37°C, 5% CO2 for 30 min. 0.01 M EDTA was added to disrupt T cell–DC complexes. .. Next, cells were depleted of CD3-expressing cells (Dynal CD3-beads; Dynal), incubated with CD11c microbeads (Miltenyi Biotec), and positively selected using MACS columns.

    Infection:

    Article Title: Resolution of a chronic viral infection after interleukin-10 receptor blockade
    Article Snippet: .. Splenocytes from mice infected 7 d earlier with LCMV Armstrong or LCMV clone 13 with or without anti–IL-10R antibody treatment were incubated with HBSS medium containing 0.5 mg/ml collagenase D (Sigma-Aldrich) at 37°C, 5% CO2 for 30 min. 0.01 M EDTA was added to disrupt T cell–DC complexes. .. Next, cells were depleted of CD3-expressing cells (Dynal CD3-beads; Dynal), incubated with CD11c microbeads (Miltenyi Biotec), and positively selected using MACS columns.

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  • 96
    Millipore matrix metalloproteinase 2
    Linear correlation between matrix <t>metalloproteinase</t> 2 (MMP-2) and aldosterone levels. In the current study, there was a significant linear relationship between MMP-2 and plasma aldosterone levels in patients with primary aldosteronism ( R =0.448, P =0.010). The Spearman correlation was used to evaluate the linear relationship between MMP-2 and aldosterone levels. APA, aldosterone-producing adenoma; BAH, bilateral adrenal hyperplasia.
    Matrix Metalloproteinase 2, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/matrix metalloproteinase 2/product/Millipore
    Average 96 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    matrix metalloproteinase 2 - by Bioz Stars, 2021-04
    96/100 stars
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    93
    Millipore recombinant human matrix metalloproteinase 2
    Macrophage migration inhibitory factor (MIF)-induced matrix metalloproteinase (MMP)-2 production is PKCδ, JNK, and Src pathway-dependent. Rheumatoid arthritis (RA) synovial fibroblasts were incubated for 6 hours, with or without MIF (50 nM), in the presence or absence of signaling pathway inhibitors: PKC (pan) inhibitor Ro-31-8425 (1 μM), PKCδ isoform-specific inhibitor rottlerin (1 μM), JNK inhibitor JNK II, and Src inhibitor PP2 (10 μM). (a) MMP-2 concentrations in cell culture supernatants were measured by ELISA. Inhibitors to PKCδ, JNK, and Src signaling intermediates inhibited MIF-induced MMP-2 upregulation. (b) Gelatin zymography showed the same effect of these inhibitors on MMP-2 upregulation. Results represent three experiments using RA synovial fibroblasts from six donors. DMSO, dimethyl sulfoxide; JNK, c-jun N-terminal kinase; NS, non-stimulated; pro-MMP, pro-matrix <t>metalloproteinase-2;</t> PKC, protein kinase C; rhMIF, recombinant human macrophage migration inhibitory factor.
    Recombinant Human Matrix Metalloproteinase 2, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/recombinant human matrix metalloproteinase 2/product/Millipore
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    93/100 stars
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    92
    Millipore matrix metalloproteinase mmp 2 antibody
    Matrix <t>metalloproteinase</t> <t>(MMP)-2</t> protein expression and activity. (Top) Western analysis detected the pro- and active form of MMP-2 in primary hepatic stellate cells. In LX cells, pro-MMP-2 was mostly detected: 40 μg of protein from cell lysates were loaded per sample. (Bottom) Gelatin zymography of conditioned media from LX-2 cells indicated secretion of pro-MMP2.
    Matrix Metalloproteinase Mmp 2 Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/matrix metalloproteinase mmp 2 antibody/product/Millipore
    Average 92 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    matrix metalloproteinase mmp 2 antibody - by Bioz Stars, 2021-04
    92/100 stars
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    94
    Millipore gelatinase b
    Effect of mMCP-4 on progelatinase B present in hyperplastic tissue and localization of mMCP-4 and <t>gelatinase</t> B in vivo. ( A ) Gelatinolytic activity in tissue lysates (2 μl) from normal (N), hyperplastic (H), dysplastic (D) and carcinoma (T) biopsies. Incubation of gelatin zymograms with 1,10 phenanthroline (an inhibitor of MMPs), but not PMSF (an inhibitor of serine proteases) following electrophoresis abolished the 68, 72, 80, and 90-kD bands completely (data not shown). ( B ) In vitro reconstitution of gelatinase B activity. Two microliters of hyperplastic (H) lysate alone or 2 μl of hyperplastic lysate incubated for 30 min at 37°C with 40 ng of purified mMCP-4. Molecular masses are shown in kD. ( C–D ) Immunolocalization of mMCP-4 (red staining) and laminin (brown staining) at basement membranes (bm, arrows) adjacent to epithelium (e) and capillaries (c) in dysplastic skin. ( E ) Immunolocalization of gelatinase B (blue staining) in dysplastic skin localizes to basement membranes (bm) adjacent to epithelium (e, closed arrows) and around capillaries (c, open arrows) in dermis. ( F ) Control for nonspecific binding using a control rabbit IgG; background staining was negligible. Bar, 44.6 μm ( C–F ).
    Gelatinase B, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 94 stars, based on 1 article reviews
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    Image Search Results


    Linear correlation between matrix metalloproteinase 2 (MMP-2) and aldosterone levels. In the current study, there was a significant linear relationship between MMP-2 and plasma aldosterone levels in patients with primary aldosteronism ( R =0.448, P =0.010). The Spearman correlation was used to evaluate the linear relationship between MMP-2 and aldosterone levels. APA, aldosterone-producing adenoma; BAH, bilateral adrenal hyperplasia.

    Journal: Endocrinology and Metabolism

    Article Title: Cardiac Dysfunction in Association with Increased Inflammatory Markers in Primary Aldosteronism

    doi: 10.3803/EnM.2016.31.4.567

    Figure Lengend Snippet: Linear correlation between matrix metalloproteinase 2 (MMP-2) and aldosterone levels. In the current study, there was a significant linear relationship between MMP-2 and plasma aldosterone levels in patients with primary aldosteronism ( R =0.448, P =0.010). The Spearman correlation was used to evaluate the linear relationship between MMP-2 and aldosterone levels. APA, aldosterone-producing adenoma; BAH, bilateral adrenal hyperplasia.

    Article Snippet: Assays for matrix metalloproteinase 2 (MMP-2) and MMP-9 were performed using the Human Cardiovascular Disease Magnetic Bead Panel (#HCVD3MAG-67K, Millipore) according to the manufacturer's protocol.

    Techniques:

    Macrophage migration inhibitory factor (MIF)-induced matrix metalloproteinase (MMP)-2 production is PKCδ, JNK, and Src pathway-dependent. Rheumatoid arthritis (RA) synovial fibroblasts were incubated for 6 hours, with or without MIF (50 nM), in the presence or absence of signaling pathway inhibitors: PKC (pan) inhibitor Ro-31-8425 (1 μM), PKCδ isoform-specific inhibitor rottlerin (1 μM), JNK inhibitor JNK II, and Src inhibitor PP2 (10 μM). (a) MMP-2 concentrations in cell culture supernatants were measured by ELISA. Inhibitors to PKCδ, JNK, and Src signaling intermediates inhibited MIF-induced MMP-2 upregulation. (b) Gelatin zymography showed the same effect of these inhibitors on MMP-2 upregulation. Results represent three experiments using RA synovial fibroblasts from six donors. DMSO, dimethyl sulfoxide; JNK, c-jun N-terminal kinase; NS, non-stimulated; pro-MMP, pro-matrix metalloproteinase-2; PKC, protein kinase C; rhMIF, recombinant human macrophage migration inhibitory factor.

    Journal: Arthritis Research & Therapy

    Article Title: Macrophage migration inhibitory factor: a mediator of matrix metalloproteinase-2 production in rheumatoid arthritis

    doi: 10.1186/ar2021

    Figure Lengend Snippet: Macrophage migration inhibitory factor (MIF)-induced matrix metalloproteinase (MMP)-2 production is PKCδ, JNK, and Src pathway-dependent. Rheumatoid arthritis (RA) synovial fibroblasts were incubated for 6 hours, with or without MIF (50 nM), in the presence or absence of signaling pathway inhibitors: PKC (pan) inhibitor Ro-31-8425 (1 μM), PKCδ isoform-specific inhibitor rottlerin (1 μM), JNK inhibitor JNK II, and Src inhibitor PP2 (10 μM). (a) MMP-2 concentrations in cell culture supernatants were measured by ELISA. Inhibitors to PKCδ, JNK, and Src signaling intermediates inhibited MIF-induced MMP-2 upregulation. (b) Gelatin zymography showed the same effect of these inhibitors on MMP-2 upregulation. Results represent three experiments using RA synovial fibroblasts from six donors. DMSO, dimethyl sulfoxide; JNK, c-jun N-terminal kinase; NS, non-stimulated; pro-MMP, pro-matrix metalloproteinase-2; PKC, protein kinase C; rhMIF, recombinant human macrophage migration inhibitory factor.

    Article Snippet: Molecular-weight marker (Sigmamarker, Sigma) and recombinant human matrix metalloproteinase-2 (rhMMP-2) were used as controls.

    Techniques: Migration, Incubation, Cell Culture, Enzyme-linked Immunosorbent Assay, Zymography, Recombinant

    Matrix metalloproteinase (MMP)-2 upregulation by macrophage migration inhibitory factor (MIF) is time-dependent. (a) Using gelatin zymography of rheumatoid arthritis (RA) synovial fibroblast culture supernatants, we found MMP-2 upregulation, beginning after 1 hour and increasing continuously over a period of 24 hours. The results represent one of four individual experiments using cells from four donors. (b) Immunofluorescence staining of RA synovial fibroblasts for MMP-2 showed a strong perinuclear and discrete diffuse cytoplasmic expression after 1 hour of stimulation by MIF (50 nM; 400×). Results represent one of four individual experiments using cells from four donors. NS, nonstimulated; pro-MMP, pro-matrix metalloproteinase-2; rhMIF, recombinant human macrophage migration inhibitory factor.

    Journal: Arthritis Research & Therapy

    Article Title: Macrophage migration inhibitory factor: a mediator of matrix metalloproteinase-2 production in rheumatoid arthritis

    doi: 10.1186/ar2021

    Figure Lengend Snippet: Matrix metalloproteinase (MMP)-2 upregulation by macrophage migration inhibitory factor (MIF) is time-dependent. (a) Using gelatin zymography of rheumatoid arthritis (RA) synovial fibroblast culture supernatants, we found MMP-2 upregulation, beginning after 1 hour and increasing continuously over a period of 24 hours. The results represent one of four individual experiments using cells from four donors. (b) Immunofluorescence staining of RA synovial fibroblasts for MMP-2 showed a strong perinuclear and discrete diffuse cytoplasmic expression after 1 hour of stimulation by MIF (50 nM; 400×). Results represent one of four individual experiments using cells from four donors. NS, nonstimulated; pro-MMP, pro-matrix metalloproteinase-2; rhMIF, recombinant human macrophage migration inhibitory factor.

    Article Snippet: Molecular-weight marker (Sigmamarker, Sigma) and recombinant human matrix metalloproteinase-2 (rhMMP-2) were used as controls.

    Techniques: Migration, Zymography, Immunofluorescence, Staining, Expressing, Recombinant

    Matrix metalloproteinase (MMP)-2 protein expression and activity. (Top) Western analysis detected the pro- and active form of MMP-2 in primary hepatic stellate cells. In LX cells, pro-MMP-2 was mostly detected: 40 μg of protein from cell lysates were loaded per sample. (Bottom) Gelatin zymography of conditioned media from LX-2 cells indicated secretion of pro-MMP2.

    Journal: Gut

    Article Title: Human hepatic stellate cell lines, LX-1 and LX-2: new tools for analysis of hepatic fibrosis

    doi: 10.1136/gut.2004.042127

    Figure Lengend Snippet: Matrix metalloproteinase (MMP)-2 protein expression and activity. (Top) Western analysis detected the pro- and active form of MMP-2 in primary hepatic stellate cells. In LX cells, pro-MMP-2 was mostly detected: 40 μg of protein from cell lysates were loaded per sample. (Bottom) Gelatin zymography of conditioned media from LX-2 cells indicated secretion of pro-MMP2.

    Article Snippet: The obese receptor long form (Ob-RL ) antibody (H300) was obtained from Santa Cruz Biotechnology, matrix metalloproteinase (MMP)-2 antibody was from Sigma-Aldrich, and MT1-MMP, tissue inhibitor of matrix metalloproteinase (TIMP)-1, and TIMP-2 were obtained from Chemicon (Temecula, California, USA).

    Techniques: Expressing, Activity Assay, Western Blot, Zymography

    Effect of mMCP-4 on progelatinase B present in hyperplastic tissue and localization of mMCP-4 and gelatinase B in vivo. ( A ) Gelatinolytic activity in tissue lysates (2 μl) from normal (N), hyperplastic (H), dysplastic (D) and carcinoma (T) biopsies. Incubation of gelatin zymograms with 1,10 phenanthroline (an inhibitor of MMPs), but not PMSF (an inhibitor of serine proteases) following electrophoresis abolished the 68, 72, 80, and 90-kD bands completely (data not shown). ( B ) In vitro reconstitution of gelatinase B activity. Two microliters of hyperplastic (H) lysate alone or 2 μl of hyperplastic lysate incubated for 30 min at 37°C with 40 ng of purified mMCP-4. Molecular masses are shown in kD. ( C–D ) Immunolocalization of mMCP-4 (red staining) and laminin (brown staining) at basement membranes (bm, arrows) adjacent to epithelium (e) and capillaries (c) in dysplastic skin. ( E ) Immunolocalization of gelatinase B (blue staining) in dysplastic skin localizes to basement membranes (bm) adjacent to epithelium (e, closed arrows) and around capillaries (c, open arrows) in dermis. ( F ) Control for nonspecific binding using a control rabbit IgG; background staining was negligible. Bar, 44.6 μm ( C–F ).

    Journal: Genes & Development

    Article Title: Inflammatory mast cells up-regulate angiogenesis during squamous epithelial carcinogenesis

    doi:

    Figure Lengend Snippet: Effect of mMCP-4 on progelatinase B present in hyperplastic tissue and localization of mMCP-4 and gelatinase B in vivo. ( A ) Gelatinolytic activity in tissue lysates (2 μl) from normal (N), hyperplastic (H), dysplastic (D) and carcinoma (T) biopsies. Incubation of gelatin zymograms with 1,10 phenanthroline (an inhibitor of MMPs), but not PMSF (an inhibitor of serine proteases) following electrophoresis abolished the 68, 72, 80, and 90-kD bands completely (data not shown). ( B ) In vitro reconstitution of gelatinase B activity. Two microliters of hyperplastic (H) lysate alone or 2 μl of hyperplastic lysate incubated for 30 min at 37°C with 40 ng of purified mMCP-4. Molecular masses are shown in kD. ( C–D ) Immunolocalization of mMCP-4 (red staining) and laminin (brown staining) at basement membranes (bm, arrows) adjacent to epithelium (e) and capillaries (c) in dysplastic skin. ( E ) Immunolocalization of gelatinase B (blue staining) in dysplastic skin localizes to basement membranes (bm) adjacent to epithelium (e, closed arrows) and around capillaries (c, open arrows) in dermis. ( F ) Control for nonspecific binding using a control rabbit IgG; background staining was negligible. Bar, 44.6 μm ( C–F ).

    Article Snippet: Skin biopsies were removed from 1-month-old transgenic mice, washed in PBS, and cut into < 1 mm2 pieces and then incubated for 24 hr at 37°C in 5% CO2 balanced air incubator in DMEM containing 0.5% BSA and either 10 m m mMCP-6, 20 m m mMCP-4, 1 ng/μl gelatinase B (Calbiochem), or medium/BSA alone.

    Techniques: In Vivo, Activity Assay, Incubation, Electrophoresis, In Vitro, Purification, Staining, Binding Assay

    A model for biphasic control of angiogenesis during squamous carcinogenesis. Skin is compartmentalized into an avascular epidermis, composed of keratinocytes and dendritic cells, and a vascularized dermis composed of fibroblasts, various hemopoietic cell types, and endothelial and smooth muscle cells forming blood vessels, embedded in a quiescent stromal milieu. During premalignant progression, stroma adjacent to neoplastic epithelium resembles that observed in chronic wounds characterized by proliferating fibroblasts, increased synthesis of α1(I) procollagen, increased vessel density, vascular permeability, increased expression and activity of ECM-degrading proteinases, increased presence of diverse leukocytes, and degranulation of MCs. MCs are exploited by neoplastic epithelia in early lesions and act to jump-start angiogenesis by their release of several bioactive molecules, e.g., bFGF, VEGF, heparin, histamine, chymase, and tryptase. Tryptase increases vascular permeability, and is a potent mitogen and activator of fibroblasts and inducer of α1(I) procollagen synthesis. Chymase, although not a direct mitogen, induces activation of angiogenesis by releasing sequestered angiogenic activity from stromal reservoirs, through gelatinase B-dependent and independent mechanisms. Gelatinase B made by reactive stromal cells, although likely involved in ECM-remodeling, releases ECM-sequestered angiogenic activity also stimulating EC chemotaxis, proliferation, and tube formation. In contrast, maintenance of neovascularization within tumor stroma is MC-independent. Sustaining angiogenesis within the cancer phase is likely accomplished directly via dramatic up-regulation of multiple heparin-binding growth factor genes in fully malignant epithelial cells.

    Journal: Genes & Development

    Article Title: Inflammatory mast cells up-regulate angiogenesis during squamous epithelial carcinogenesis

    doi:

    Figure Lengend Snippet: A model for biphasic control of angiogenesis during squamous carcinogenesis. Skin is compartmentalized into an avascular epidermis, composed of keratinocytes and dendritic cells, and a vascularized dermis composed of fibroblasts, various hemopoietic cell types, and endothelial and smooth muscle cells forming blood vessels, embedded in a quiescent stromal milieu. During premalignant progression, stroma adjacent to neoplastic epithelium resembles that observed in chronic wounds characterized by proliferating fibroblasts, increased synthesis of α1(I) procollagen, increased vessel density, vascular permeability, increased expression and activity of ECM-degrading proteinases, increased presence of diverse leukocytes, and degranulation of MCs. MCs are exploited by neoplastic epithelia in early lesions and act to jump-start angiogenesis by their release of several bioactive molecules, e.g., bFGF, VEGF, heparin, histamine, chymase, and tryptase. Tryptase increases vascular permeability, and is a potent mitogen and activator of fibroblasts and inducer of α1(I) procollagen synthesis. Chymase, although not a direct mitogen, induces activation of angiogenesis by releasing sequestered angiogenic activity from stromal reservoirs, through gelatinase B-dependent and independent mechanisms. Gelatinase B made by reactive stromal cells, although likely involved in ECM-remodeling, releases ECM-sequestered angiogenic activity also stimulating EC chemotaxis, proliferation, and tube formation. In contrast, maintenance of neovascularization within tumor stroma is MC-independent. Sustaining angiogenesis within the cancer phase is likely accomplished directly via dramatic up-regulation of multiple heparin-binding growth factor genes in fully malignant epithelial cells.

    Article Snippet: Skin biopsies were removed from 1-month-old transgenic mice, washed in PBS, and cut into < 1 mm2 pieces and then incubated for 24 hr at 37°C in 5% CO2 balanced air incubator in DMEM containing 0.5% BSA and either 10 m m mMCP-6, 20 m m mMCP-4, 1 ng/μl gelatinase B (Calbiochem), or medium/BSA alone.

    Techniques: Permeability, Expressing, Activity Assay, Activated Clotting Time Assay, Activation Assay, Chemotaxis Assay, Binding Assay