collagenase d  (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 collagenase d
    Collagenase D
    Collagenase D is prepared from Clostridium histolyticum cultures by filtration ammonium sulfate precipitation dialysis and lyophilization
    https://www.bioz.com/result/collagenase d/product/Millipore
    Average 99 stars, based on 29 article reviews
    Price from $9.99 to $1999.99
    collagenase d - by Bioz Stars, 2021-01
    99/100 stars

    Images

    1) Product Images from "Polarization of macrophages in the tumor microenvironment is influenced by EGFR signaling within colon cancer cells"

    Article Title: Polarization of macrophages in the tumor microenvironment is influenced by EGFR signaling within colon cancer cells

    Journal: Oncotarget

    doi: 10.18632/oncotarget.12207

    Cetuximab modulates macrophage polarization in an AOM/DSS mouse model A. Establishment of the AOM/DSS mouse model. AOM was injected intraperitoneally at 12.5 mg/kg body weight. After one week, mice were given drinking water containing 2.5% DSS for 5 days followed by 16 days of regular drinking water. After two cycles of DSS treatment, cetuximab (1 mg/mouse, twice a week) was injected intraperitoneally for a month, and the mice were then sacrificed. B. Representative images of colon tumors in normal (right), AOM/DSS (2AD) (middle), and cetuximab-treated AOM/DSS mice (2AD+cetu) (left). C. Tumor quantification. Cetuximab treatment (2AD + cetu) reduced tumor numbers compared to 2AD mice. D. p-EGFR (Y1068), EGFR, Arg1, and iNOS protein levels in normal mice, 2AD and 2AD+cetu mouse tumors were detected by Western blot. All experiments were repeated three times. E. Representative photomicrographs of immunostaining for p-EGFR (Y1068), PCNA, F4/80, Arg1, and iNOS. Scale bars: 25 μm. F. M1 marker (IL-12, iNOS) and M2 marker (IL-4, IL-10, Arg1) mRNA levels in normal mouse colon tissues, 2AD and 2AD+cetu mouse tumor tissues were evaluated by q-PCR. G. Percentages of CD11b + /F4/80 + and F4/80 + /CD206 + cells in normal, 2AD, and 2AD+cetu mice colon tissues were detected by flow cytometry. Colon tissues were cut into small pieces (1-2 mm) and incubated with collagenase D (1- mg/mL), dispase II (1 mg/mL), and DNase I (100 μg/mL) for 30-45 min in a shaking incubator at 37°C, and single-cell suspensions were then incubated with antibodies. Bars represent means ± SD (n = 3) for each treatment. * p
    Figure Legend Snippet: Cetuximab modulates macrophage polarization in an AOM/DSS mouse model A. Establishment of the AOM/DSS mouse model. AOM was injected intraperitoneally at 12.5 mg/kg body weight. After one week, mice were given drinking water containing 2.5% DSS for 5 days followed by 16 days of regular drinking water. After two cycles of DSS treatment, cetuximab (1 mg/mouse, twice a week) was injected intraperitoneally for a month, and the mice were then sacrificed. B. Representative images of colon tumors in normal (right), AOM/DSS (2AD) (middle), and cetuximab-treated AOM/DSS mice (2AD+cetu) (left). C. Tumor quantification. Cetuximab treatment (2AD + cetu) reduced tumor numbers compared to 2AD mice. D. p-EGFR (Y1068), EGFR, Arg1, and iNOS protein levels in normal mice, 2AD and 2AD+cetu mouse tumors were detected by Western blot. All experiments were repeated three times. E. Representative photomicrographs of immunostaining for p-EGFR (Y1068), PCNA, F4/80, Arg1, and iNOS. Scale bars: 25 μm. F. M1 marker (IL-12, iNOS) and M2 marker (IL-4, IL-10, Arg1) mRNA levels in normal mouse colon tissues, 2AD and 2AD+cetu mouse tumor tissues were evaluated by q-PCR. G. Percentages of CD11b + /F4/80 + and F4/80 + /CD206 + cells in normal, 2AD, and 2AD+cetu mice colon tissues were detected by flow cytometry. Colon tissues were cut into small pieces (1-2 mm) and incubated with collagenase D (1- mg/mL), dispase II (1 mg/mL), and DNase I (100 μg/mL) for 30-45 min in a shaking incubator at 37°C, and single-cell suspensions were then incubated with antibodies. Bars represent means ± SD (n = 3) for each treatment. * p

    Techniques Used: Injection, Mouse Assay, Western Blot, Immunostaining, Marker, Polymerase Chain Reaction, Flow Cytometry, Cytometry, Incubation

    2) Product Images from "Galectin-3 captures interferon-gamma in the tumor matrix reducing chemokine gradient production and T-cell tumor infiltration"

    Article Title: Galectin-3 captures interferon-gamma in the tumor matrix reducing chemokine gradient production and T-cell tumor infiltration

    Journal: Nature Communications

    doi: 10.1038/s41467-017-00925-6

    Spreading of IFNγ signaling in tumor xenografts treated in vivo with collagenase D. a CXCL9 fold induction along the tumor sections in mice treated with IFNγ alone (50 ng per tumor) or together with collagenase D (2.5 μg per tumor). Mean ± SEM of six independent tumors for each treatment. For CXCL9 induction in individual tumors see Supplementary Fig. 6b . *** P = 0.0006; Paired t -test, t = 4.035 and degree of freedom = 20. The red dotted line marks the threshold of 50 CXCL9 mRNA copies per section of the tumor. Each section represents 0.66 mm thick, contains 30 tumor slices, and about 1–4 millions cells. The drawing below shows the diffusion of CXCL9 gradient along the tumor taking into account the threshold. b Quantification of CXCL9 staining area in immunohistochemistry images of several sections of tumors treated either with IFNγ alone or combined with collagenase D. Representative images are shown in Supplementary Fig. 8
    Figure Legend Snippet: Spreading of IFNγ signaling in tumor xenografts treated in vivo with collagenase D. a CXCL9 fold induction along the tumor sections in mice treated with IFNγ alone (50 ng per tumor) or together with collagenase D (2.5 μg per tumor). Mean ± SEM of six independent tumors for each treatment. For CXCL9 induction in individual tumors see Supplementary Fig. 6b . *** P = 0.0006; Paired t -test, t = 4.035 and degree of freedom = 20. The red dotted line marks the threshold of 50 CXCL9 mRNA copies per section of the tumor. Each section represents 0.66 mm thick, contains 30 tumor slices, and about 1–4 millions cells. The drawing below shows the diffusion of CXCL9 gradient along the tumor taking into account the threshold. b Quantification of CXCL9 staining area in immunohistochemistry images of several sections of tumors treated either with IFNγ alone or combined with collagenase D. Representative images are shown in Supplementary Fig. 8

    Techniques Used: In Vivo, Mouse Assay, Diffusion-based Assay, Staining, Immunohistochemistry

    3) Product Images from "Resolution of a chronic viral infection after interleukin-10 receptor blockade"

    Article Title: Resolution of a chronic viral infection after interleukin-10 receptor blockade

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20061462

    Anti–IL-10R treatment restores cell numbers in LCMV clone 13–infected mice and resets the CD8α −  versus CD8α +  DC ratio to levels observed in LCMV Armstrong–infected mice.  (A) The effect of 250 μg anti–IL-10R antibody (i.p.) or IgG 1  isotype control antibody treatment on different splenic cell subsets was monitored quantitatively. Spleens of mice infected 3 or 21 wk earlier with LCMV clone 13 were harvested, and cell suspensions were stained with fluorescent antibodies to CD4, CD8, B220, CD11c, and CD11b. Age-matched naive mice were included as controls. Numbers of cells were quantified by relating the percentage to total numbers of spleen cells. Mean values for three to five individual mice are shown. Results are representative of three similar experiments. (B and C) The total numbers of CD11c + CD8α −  (B, top) or CD11c + CD8α +  splenic DC subsets (B, bottom) and the CD8α −  to CD8α +  DC ratios (C) were determined 0, 1, 2, and 6 wk after LCMV Armstrong or LCMV clone 13 infection. In addition, absolute splenic DC subsets (B) and the CD8α −  to CD8α +  DC ratio (C) were calculated from mice therapeutically treated with anti–IL10R on days 7 and 14 after LCMV clone 13 infection; the latter was examined at 6 wk after infection only. Cell suspensions were prepared by digestion with collagenase D. Cells were then stained with fluorescent antibodies to CD11c, CD3, and CD8α. The percentage of CD8α −  and CD8α +  within the CD11c + CD3 −  subset was determined by flow cytometry, and total numbers per spleen were quantified by relating the percentage of these cells to total numbers of CD11c + CD3 −  spleen cells. Histogram bars represent mean values ± SD for three mice per group. The experiment is representative of three similar experiments. Statistical analysis was performed using the Student's  t  test. *, P
    Figure Legend Snippet: Anti–IL-10R treatment restores cell numbers in LCMV clone 13–infected mice and resets the CD8α − versus CD8α + DC ratio to levels observed in LCMV Armstrong–infected mice. (A) The effect of 250 μg anti–IL-10R antibody (i.p.) or IgG 1 isotype control antibody treatment on different splenic cell subsets was monitored quantitatively. Spleens of mice infected 3 or 21 wk earlier with LCMV clone 13 were harvested, and cell suspensions were stained with fluorescent antibodies to CD4, CD8, B220, CD11c, and CD11b. Age-matched naive mice were included as controls. Numbers of cells were quantified by relating the percentage to total numbers of spleen cells. Mean values for three to five individual mice are shown. Results are representative of three similar experiments. (B and C) The total numbers of CD11c + CD8α − (B, top) or CD11c + CD8α + splenic DC subsets (B, bottom) and the CD8α − to CD8α + DC ratios (C) were determined 0, 1, 2, and 6 wk after LCMV Armstrong or LCMV clone 13 infection. In addition, absolute splenic DC subsets (B) and the CD8α − to CD8α + DC ratio (C) were calculated from mice therapeutically treated with anti–IL10R on days 7 and 14 after LCMV clone 13 infection; the latter was examined at 6 wk after infection only. Cell suspensions were prepared by digestion with collagenase D. Cells were then stained with fluorescent antibodies to CD11c, CD3, and CD8α. The percentage of CD8α − and CD8α + within the CD11c + CD3 − subset was determined by flow cytometry, and total numbers per spleen were quantified by relating the percentage of these cells to total numbers of CD11c + CD3 − spleen cells. Histogram bars represent mean values ± SD for three mice per group. The experiment is representative of three similar experiments. Statistical analysis was performed using the Student's t test. *, P

    Techniques Used: Infection, Mouse Assay, Staining, Flow Cytometry, Cytometry

    Related Articles

    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).

    Isolation:

    Article Title: Analysis of Tks4 Knockout Mice Suggests a Role for Tks4 in Adipose Tissue Homeostasis in the Context of Beigeing
    Article Snippet: .. Cell Isolation, Analysis, and Culture 10–14-week-old Tks4−/− and WT mice were euthanized, and sWAT, gWAT, and iBAT fat pads were isolated, minced, and incubated in digestive medium containing 0.1% (m /v ) (for sWAT and gWAT) or 0.2% (m /v ) (for iBAT) type IV collagenase in Dulbecco’s phosphate-buffered saline (all from Sigma-Aldrich) for 1 h at 37 °C on a shaker. .. The tissue digestion was stopped by adding an equal volume of fetal bovine serum (FBS), and the resulting cell suspensions were centrifuged twice at 300× g for 5 min in Hank’s balanced salt solution (HBSS) (all from Invitrogen, Carlsbad, CA, USA) to isolate the stromal vascular fraction (SVF).

    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. ..

    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).

    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.

    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.

    Article Title: Analysis of Tks4 Knockout Mice Suggests a Role for Tks4 in Adipose Tissue Homeostasis in the Context of Beigeing
    Article Snippet: .. Cell Isolation, Analysis, and Culture 10–14-week-old Tks4−/− and WT mice were euthanized, and sWAT, gWAT, and iBAT fat pads were isolated, minced, and incubated in digestive medium containing 0.1% (m /v ) (for sWAT and gWAT) or 0.2% (m /v ) (for iBAT) type IV collagenase in Dulbecco’s phosphate-buffered saline (all from Sigma-Aldrich) for 1 h at 37 °C on a shaker. .. The tissue digestion was stopped by adding an equal volume of fetal bovine serum (FBS), and the resulting cell suspensions were centrifuged twice at 300× g for 5 min in Hank’s balanced salt solution (HBSS) (all from Invitrogen, Carlsbad, CA, USA) to isolate the stromal vascular fraction (SVF).

    Concentration Assay:

    Article Title: Characterisation of gut, lung, and upper airways microbiota in patients with non-small cell lung carcinoma
    Article Snippet: .. The homogenate obtained will be treated with collagenase D (Sigma-Aldrich) (2 mg/mL final concentration) at 37°C for 15 min, followed by 10 min at 2000 × g at room temperature (RT). .. The pellet will be resuspended in 2–5 mL (depending on the initial sample weight) of mammalian cell lysis buffer (MCLB),[ ] and repeatedly vortexed for 5 min at RT.

    Incubation:

    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.

    Article Title: Analysis of Tks4 Knockout Mice Suggests a Role for Tks4 in Adipose Tissue Homeostasis in the Context of Beigeing
    Article Snippet: .. Cell Isolation, Analysis, and Culture 10–14-week-old Tks4−/− and WT mice were euthanized, and sWAT, gWAT, and iBAT fat pads were isolated, minced, and incubated in digestive medium containing 0.1% (m /v ) (for sWAT and gWAT) or 0.2% (m /v ) (for iBAT) type IV collagenase in Dulbecco’s phosphate-buffered saline (all from Sigma-Aldrich) for 1 h at 37 °C on a shaker. .. The tissue digestion was stopped by adding an equal volume of fetal bovine serum (FBS), and the resulting cell suspensions were centrifuged twice at 300× g for 5 min in Hank’s balanced salt solution (HBSS) (all from Invitrogen, Carlsbad, CA, USA) to isolate the stromal vascular fraction (SVF).

    Article Title: Polarization of macrophages in the tumor microenvironment is influenced by EGFR signaling within colon cancer cells
    Article Snippet: .. 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.

    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).

    Cell Isolation:

    Article Title: Analysis of Tks4 Knockout Mice Suggests a Role for Tks4 in Adipose Tissue Homeostasis in the Context of Beigeing
    Article Snippet: .. Cell Isolation, Analysis, and Culture 10–14-week-old Tks4−/− and WT mice were euthanized, and sWAT, gWAT, and iBAT fat pads were isolated, minced, and incubated in digestive medium containing 0.1% (m /v ) (for sWAT and gWAT) or 0.2% (m /v ) (for iBAT) type IV collagenase in Dulbecco’s phosphate-buffered saline (all from Sigma-Aldrich) for 1 h at 37 °C on a shaker. .. The tissue digestion was stopped by adding an equal volume of fetal bovine serum (FBS), and the resulting cell suspensions were centrifuged twice at 300× g for 5 min in Hank’s balanced salt solution (HBSS) (all from Invitrogen, Carlsbad, CA, USA) to isolate the stromal vascular fraction (SVF).

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