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Bio-Rad polyclonal rabbit anti murine mmp 9 igg
Polyclonal Rabbit Anti Murine Mmp 9 Igg, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 91/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/polyclonal+rabbit+anti+murine+mmp+9+igg/Rabbit+anti+MMP-2/pmc07347292-208-20-42
Average 91 stars, based on 12 article reviews

polyclonal rabbit anti murine mmp 9 igg - by Bioz Stars, 2026-10

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Electrophoresis:

Article Title: Tissue Inhibitor of Metalloproteinase-1 (TIMP-1) Promotes PMN Pericellular Proteolysis by Anchoring Matrix Metalloproteinase-8 and -9 to PMN Surfaces
Article Snippet: .. Briefly, samples were subjected to electrophoresis on 8% SDS-PAGE under reducing conditions, proteins transferred to PVDF membranes, and probed with polyclonal rabbit anti-murine Mmp-9 IgG (AB19047) or rabbit anti-murine Mmp-8 IgG (Ab19045), followed by goat anti-rabbit IgG conjugated to horse radish peroxidase (Biorad, Hercules, CA), and a chemiluminescence detection system following the manufacturer’s instructions. ..

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Image Search Results

Human PMNs were activated at 37°C with PAF at 10−7 M for 15 min followed by fMLP at 10−7 M for 30 min. Cells were double immunostained with Alexa 546 and murine anti-MMP-9 IgG (A, second panel), or murine anti-human MMP-8 IgG (B, second panel) or non-immune murine (Ms) IgG (C, second panel) and with Alexa 488 and rabbit anti-TIMP-1 IgG (A and B third panels) or non-immune rabbit (Rb) IgG (C, third panel). The anti-MMP-8 and anti-MMP-9 IgGs used recognize both pro and active forms of these MMPs. Cells were examined using a Normarski objective (A-C, left panels) and co-localization of MMPs and TIMP-1 on the surface of the activated PMNs was assessed by confocal microscopy (see merged images in the right panels for A-C). The white bars are 5 microns in length. Note the strong co-localization of TIMP-1 with both MMP-8 and MMP-9 on the surface of activated PMNs. The results shown are representative of at least 3 different PMN preparations.

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Tissue Inhibitor of Metalloproteinase-1 (TIMP-1) Promotes PMN Pericellular Proteolysis by Anchoring Matrix Metalloproteinase-8 and -9 to PMN Surfaces

doi: 10.4049/jimmunol.1801466

Figure Lengend Snippet: Human PMNs were activated at 37°C with PAF at 10−7 M for 15 min followed by fMLP at 10−7 M for 30 min. Cells were double immunostained with Alexa 546 and murine anti-MMP-9 IgG (A, second panel), or murine anti-human MMP-8 IgG (B, second panel) or non-immune murine (Ms) IgG (C, second panel) and with Alexa 488 and rabbit anti-TIMP-1 IgG (A and B third panels) or non-immune rabbit (Rb) IgG (C, third panel). The anti-MMP-8 and anti-MMP-9 IgGs used recognize both pro and active forms of these MMPs. Cells were examined using a Normarski objective (A-C, left panels) and co-localization of MMPs and TIMP-1 on the surface of the activated PMNs was assessed by confocal microscopy (see merged images in the right panels for A-C). The white bars are 5 microns in length. Note the strong co-localization of TIMP-1 with both MMP-8 and MMP-9 on the surface of activated PMNs. The results shown are representative of at least 3 different PMN preparations.

Article Snippet: Polyclonal rabbit anti-human MMP-9 IgG (AB805), polyclonal rabbit anti-murine Mmp-9 IgG (AB19047), murine anti-human MMP-9 IgG (MAB13416), polyclonal rabbit anti-human MMP-8 IgG (AB8115), polyclonal rabbit anti-TIMP-1 IgG (AB800), and rabbit anti-TIMP-2 IgG (AB771) were purchased from Millipore (Billerica, MA).

Techniques: Confocal Microscopy

In A and B, PMNs from Mmp-8−/− x Mmp-9−/− mice were activated for 15 min at 37°C with 10−6 M PAF followed by 10−6 M fMLP to induce surface expression of Timp-1. In A, the activated PMNs were then pre-incubated for 45 min at 4°C with or without exogenous full length murine proMmp-8 (50–400 nM). Cells were then incubated for an additional 60 min at 4°C with 100 nM exogenous murine proMMP-9. Cells were then washed and immunostained with rabbit anti-murine Mmp-9 IgG, or non-immune rabbit IgG followed by goat anti-rabbit F(ab)2-conjugated to Alexa 488 to detect bound pro and active Mmp-9. In B, the PAF- and fMLP- activated and PMNs were pre-incubated for 45 min at 4°C with or without exogenous full length murine proMmp-9 (50–400 nM). Cells were then incubated for an additional 60 min at 4°C with 100 nM exogenous full length murine proMMP-8. Cells were then immunostained with rabbit anti-murine Mmp-8 IgG, or non-immune rabbit IgG followed by goat anti-rabbit F(ab)2-conjugated to Alexa 488 to detect bound pro and active Mmp-8. In A and B, images of immunostained cells were captured and surface-bound pro and active Mmp-9 (in A) or pro and active Mmp-8 (in B) were quantified using MetaMorph software. Data are mean + SEM (n = 150–200 cells/group). Data were analyzed using a One-Way ANOVA followed by pair-wise testing with two-tailed Student’s t-tests. Asterisk indicates P < 0.001 when compared with PMNs incubated in the absence of exogenous Mmp. The results shown are representative of at least 3 independent experiments.

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Tissue Inhibitor of Metalloproteinase-1 (TIMP-1) Promotes PMN Pericellular Proteolysis by Anchoring Matrix Metalloproteinase-8 and -9 to PMN Surfaces

doi: 10.4049/jimmunol.1801466

Figure Lengend Snippet: In A and B, PMNs from Mmp-8−/− x Mmp-9−/− mice were activated for 15 min at 37°C with 10−6 M PAF followed by 10−6 M fMLP to induce surface expression of Timp-1. In A, the activated PMNs were then pre-incubated for 45 min at 4°C with or without exogenous full length murine proMmp-8 (50–400 nM). Cells were then incubated for an additional 60 min at 4°C with 100 nM exogenous murine proMMP-9. Cells were then washed and immunostained with rabbit anti-murine Mmp-9 IgG, or non-immune rabbit IgG followed by goat anti-rabbit F(ab)2-conjugated to Alexa 488 to detect bound pro and active Mmp-9. In B, the PAF- and fMLP- activated and PMNs were pre-incubated for 45 min at 4°C with or without exogenous full length murine proMmp-9 (50–400 nM). Cells were then incubated for an additional 60 min at 4°C with 100 nM exogenous full length murine proMMP-8. Cells were then immunostained with rabbit anti-murine Mmp-8 IgG, or non-immune rabbit IgG followed by goat anti-rabbit F(ab)2-conjugated to Alexa 488 to detect bound pro and active Mmp-8. In A and B, images of immunostained cells were captured and surface-bound pro and active Mmp-9 (in A) or pro and active Mmp-8 (in B) were quantified using MetaMorph software. Data are mean + SEM (n = 150–200 cells/group). Data were analyzed using a One-Way ANOVA followed by pair-wise testing with two-tailed Student’s t-tests. Asterisk indicates P < 0.001 when compared with PMNs incubated in the absence of exogenous Mmp. The results shown are representative of at least 3 independent experiments.

Techniques: Expressing, Incubation, Software, Two Tailed Test

In A and B, PMNs from WT, Timp-1−/−, Mmp-8−/−, and Mmp-9−/− mice were activated for 15 min at 37°C with 10−6 M PAF followed by 10−6 M fMLP. The cells were then fixed and immunostained for surface-bound Mmp-8 (in A) or surface-bound Mmp-9 (in B) using antibodies that detect both pro and active forms of these Mmps. Surface levels of Mmp-8 or Mmp-9 were quantified as described in Methods and expressed as a percentage of the amount of Mmp-8 or Mmp-9 respectively, that were expressed on the surface of activated WT PMNs. Data are mean + SEM from 3–4 separate experiments. Data were analyzed using a One-Way ANOVA followed by pair-wise testing with two-tailed Student’s t-tests. Asterisk indicates P < 0.01 compared with WT PMNs. In C-E, PMNs from WT, Timp-1−/−, and Mmp-8−/− and or Mmp-9−/− mice were activated and fixed as described in A. Equal numbers of cells from each genotype were pre-incubated for 1 h at 37°C in triplicate with and without 1–10-phenanthroline (a non-selective MMP inhibitor). Cells were then incubated at 37°C with: 1) type I-collagen conjugated to quenched FITC for 18 h (in C); 2) gelatin conjugated to quenched FITC for 6 h (in D); or 3) McaPLGLDpaAR (a quenched fluorogenic peptide substrate which is cleaved by both Mmp-8 and Mmp-9) for 3 h (in E). Mmp-mediated cleavage of each substrate was quantified in cell-free supernatant samples by fluorimetry as the 1,10-phenanthroline-inhibitable cleavage of the substrate, as described in Methods. The data are expressed as mean + SEM % cleavage of the substrate by WT cells from 4–5 separate experiments. Data were analyzed using a One-Way ANOVA followed by pair-wise testing with two-tailed Student’s t-tests. Asterisk indicates P < 0.01 compared with WT PMNs or the group indicated.

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Tissue Inhibitor of Metalloproteinase-1 (TIMP-1) Promotes PMN Pericellular Proteolysis by Anchoring Matrix Metalloproteinase-8 and -9 to PMN Surfaces

doi: 10.4049/jimmunol.1801466

Figure Lengend Snippet: In A and B, PMNs from WT, Timp-1−/−, Mmp-8−/−, and Mmp-9−/− mice were activated for 15 min at 37°C with 10−6 M PAF followed by 10−6 M fMLP. The cells were then fixed and immunostained for surface-bound Mmp-8 (in A) or surface-bound Mmp-9 (in B) using antibodies that detect both pro and active forms of these Mmps. Surface levels of Mmp-8 or Mmp-9 were quantified as described in Methods and expressed as a percentage of the amount of Mmp-8 or Mmp-9 respectively, that were expressed on the surface of activated WT PMNs. Data are mean + SEM from 3–4 separate experiments. Data were analyzed using a One-Way ANOVA followed by pair-wise testing with two-tailed Student’s t-tests. Asterisk indicates P < 0.01 compared with WT PMNs. In C-E, PMNs from WT, Timp-1−/−, and Mmp-8−/− and or Mmp-9−/− mice were activated and fixed as described in A. Equal numbers of cells from each genotype were pre-incubated for 1 h at 37°C in triplicate with and without 1–10-phenanthroline (a non-selective MMP inhibitor). Cells were then incubated at 37°C with: 1) type I-collagen conjugated to quenched FITC for 18 h (in C); 2) gelatin conjugated to quenched FITC for 6 h (in D); or 3) McaPLGLDpaAR (a quenched fluorogenic peptide substrate which is cleaved by both Mmp-8 and Mmp-9) for 3 h (in E). Mmp-mediated cleavage of each substrate was quantified in cell-free supernatant samples by fluorimetry as the 1,10-phenanthroline-inhibitable cleavage of the substrate, as described in Methods. The data are expressed as mean + SEM % cleavage of the substrate by WT cells from 4–5 separate experiments. Data were analyzed using a One-Way ANOVA followed by pair-wise testing with two-tailed Student’s t-tests. Asterisk indicates P < 0.01 compared with WT PMNs or the group indicated.

Techniques: Two Tailed Test, Incubation

Mmp-9 protein levels contained within and released by WT and Timp-1 −/− PMNs

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Tissue Inhibitor of Metalloproteinase-1 (TIMP-1) Promotes PMN Pericellular Proteolysis by Anchoring Matrix Metalloproteinase-8 and -9 to PMN Surfaces

doi: 10.4049/jimmunol.1801466

Figure Lengend Snippet: Mmp-9 protein levels contained within and released by WT and Timp-1 −/− PMNs

Techniques:

Extracts of freshly-isolated unstimulated PMNs from WT and Timp-1−/− mice and cell-free supernatant fluids from WT and Timp-1−/− PMN that had been activated with 10−6 M fMLP for 30 min were immuno-blotted for Mmp-9 (A) or Mmp-8 (B). The arrows indicate the proMMP-9 and proMmp-8 forms of the Mmps. Note that extracts of unstimulated WT and Timp-1−/− PMNs contain similar amounts of proMmp-9 and proMmp-8 (Mr = 100 kDa for Mmp-9 and 85 kDa for Mmp-8). In addition, when activated with fMLP, Timp-1−/− PMNs freely release similar amounts of Mmp-8 and Mmp-9 exclusively as the latent proMmp forms.

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Tissue Inhibitor of Metalloproteinase-1 (TIMP-1) Promotes PMN Pericellular Proteolysis by Anchoring Matrix Metalloproteinase-8 and -9 to PMN Surfaces

doi: 10.4049/jimmunol.1801466

Figure Lengend Snippet: Extracts of freshly-isolated unstimulated PMNs from WT and Timp-1−/− mice and cell-free supernatant fluids from WT and Timp-1−/− PMN that had been activated with 10−6 M fMLP for 30 min were immuno-blotted for Mmp-9 (A) or Mmp-8 (B). The arrows indicate the proMMP-9 and proMmp-8 forms of the Mmps. Note that extracts of unstimulated WT and Timp-1−/− PMNs contain similar amounts of proMmp-9 and proMmp-8 (Mr = 100 kDa for Mmp-9 and 85 kDa for Mmp-8). In addition, when activated with fMLP, Timp-1−/− PMNs freely release similar amounts of Mmp-8 and Mmp-9 exclusively as the latent proMmp forms.

Techniques: Isolation

In A, PAF- and fMLP-activated PMNs from Mmp-9−/− and Timp-1−/− mice were incubated with or without 50–400 nM exogenous murine proMmp-9, and then immunostained with rabbit anti-Mmp-9 IgG, or non-immune rabbit IgG followed by goat anti-rabbit F(ab)2-conjugated to Alexa 488. In B, PAF- and fMLP-activated PMNs from Mmp-8−/− and Timp-1−/− mice were incubated with or without 50–400 nM exogenous murine proMmp-8, and then immunostained with rabbit anti-Mmp-8 IgG, or non-immune rabbit IgG followed by goat- anti-rabbit F(ab)2-conjugated to Alexa 488. In A-B, the antibodies used detect both the pro and active forms of the Mmps. Data are mean + SEM; n = 150–200 cells per group. Data were analyzed using a One-Way ANOVA followed by pair-wise testing with two-tailed Student’s t-tests. Asterisk indicates P < 0.001 compared with cells incubated without exogenous murine proMmp-9 or proMmp-8. In C, Timp-1−/− PMNs were incubated at 4°C with: 1) no exogenous proteins for 2 h; 2) no exogenous proteins for 1 h and then 200 nM proMmp-9 for 1 h; 3) 200 nM Timp-1 for 1 h and then with proMmp-9 for 1 h; 4) 200 nM Timp-2 for 1 h, and then with proMmp-9 for 1 h. Unbound proteins were removed after each incubation step by washing PMN twice in buffer. Non-permeabilized PMNs were then immunostained with Alexa 488 for surface-bound pro and active Mmp-9. Data are mean + SEM; n = 150–200 cells per group. Data were analyzed using a One-Way ANOVA followed by pair-wise testing with two-tailed Student’s t-tests. Asterisk indicates P < 0.001 compared with all other groups. In D, Timp-1−/− PMN were incubated at 4°C with: 1) no exogenous proteins for 2 h; 2) no exogenous proteins for 1 h and then 200 nM proMmp-8 for 1 h; 3) 200 nM Timp-1 for 1 h and then proMmp-8 for 1 h; 4) 200 nM Timp-2 for 1 h and then proMmp-8 for 1 h; 5) 200 nM myeloperoxidase for 1 h and then 200 nM proMmp-8 for 1 h. Unbound proteins were removed after each incubation step by washing PMN twice in PBS. Non-permeabilized PMNs were then immunostained with Alexa 488 for surface-bound pro and active Mmp-8. Data are mean + SEM; n = 150–200 cells per group. Data were analyzed using a One-Way ANOVA followed by pair-wise testing with two-tailed Student’s t-tests. Asterisk indicates P < 0.001 compared with all other groups. In A-D, the results shown are representative of at least 3 independent experiments. In E-F, equal numbers of PAF- and fMLP-activated PMNs from Timp-1−/− mice were incubated at 4°C with: 1) no exogenous proteins for 2 h; 2) no exogenous proteins for 1 h and 200 nM proMmp-9 or proMmp-8 alone for 1 h; 3) 200 nM Timp-1 for 1 h and then 200 nM proMmp-9 or proMmp-8 for 1 h; 4) 200 nM Timp-2 for 1 h and then 200 nM proMmp-9 or proMmp-8 for 1 h; The cells were fixed and then incubated at 37°C with: 1) gelatin conjugated to quenched FITC for 18 h (in E); or 2) type-I collagen conjugated to quenched FITC for 18 h (in F). In E-F, Mmp-mediated cleavage of each substrate in the presence of amino-phenyl mercuric acetate was quantified in cell-free supernatant samples using fluorimetry, as described in Methods. The data are expressed as mean + SEM % cleavage of the substrate in cells incubated without exogenous proteins; n=4–5 separate experiments. Data were analyzed using a Kruskal-Wallis One-Way ANOVA followed by pair-wise testing with Mann-Whitney U tests. Asterisks indicate P < 0.05 compared with all other groups.

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Tissue Inhibitor of Metalloproteinase-1 (TIMP-1) Promotes PMN Pericellular Proteolysis by Anchoring Matrix Metalloproteinase-8 and -9 to PMN Surfaces

doi: 10.4049/jimmunol.1801466

Figure Lengend Snippet: In A, PAF- and fMLP-activated PMNs from Mmp-9−/− and Timp-1−/− mice were incubated with or without 50–400 nM exogenous murine proMmp-9, and then immunostained with rabbit anti-Mmp-9 IgG, or non-immune rabbit IgG followed by goat anti-rabbit F(ab)2-conjugated to Alexa 488. In B, PAF- and fMLP-activated PMNs from Mmp-8−/− and Timp-1−/− mice were incubated with or without 50–400 nM exogenous murine proMmp-8, and then immunostained with rabbit anti-Mmp-8 IgG, or non-immune rabbit IgG followed by goat- anti-rabbit F(ab)2-conjugated to Alexa 488. In A-B, the antibodies used detect both the pro and active forms of the Mmps. Data are mean + SEM; n = 150–200 cells per group. Data were analyzed using a One-Way ANOVA followed by pair-wise testing with two-tailed Student’s t-tests. Asterisk indicates P < 0.001 compared with cells incubated without exogenous murine proMmp-9 or proMmp-8. In C, Timp-1−/− PMNs were incubated at 4°C with: 1) no exogenous proteins for 2 h; 2) no exogenous proteins for 1 h and then 200 nM proMmp-9 for 1 h; 3) 200 nM Timp-1 for 1 h and then with proMmp-9 for 1 h; 4) 200 nM Timp-2 for 1 h, and then with proMmp-9 for 1 h. Unbound proteins were removed after each incubation step by washing PMN twice in buffer. Non-permeabilized PMNs were then immunostained with Alexa 488 for surface-bound pro and active Mmp-9. Data are mean + SEM; n = 150–200 cells per group. Data were analyzed using a One-Way ANOVA followed by pair-wise testing with two-tailed Student’s t-tests. Asterisk indicates P < 0.001 compared with all other groups. In D, Timp-1−/− PMN were incubated at 4°C with: 1) no exogenous proteins for 2 h; 2) no exogenous proteins for 1 h and then 200 nM proMmp-8 for 1 h; 3) 200 nM Timp-1 for 1 h and then proMmp-8 for 1 h; 4) 200 nM Timp-2 for 1 h and then proMmp-8 for 1 h; 5) 200 nM myeloperoxidase for 1 h and then 200 nM proMmp-8 for 1 h. Unbound proteins were removed after each incubation step by washing PMN twice in PBS. Non-permeabilized PMNs were then immunostained with Alexa 488 for surface-bound pro and active Mmp-8. Data are mean + SEM; n = 150–200 cells per group. Data were analyzed using a One-Way ANOVA followed by pair-wise testing with two-tailed Student’s t-tests. Asterisk indicates P < 0.001 compared with all other groups. In A-D, the results shown are representative of at least 3 independent experiments. In E-F, equal numbers of PAF- and fMLP-activated PMNs from Timp-1−/− mice were incubated at 4°C with: 1) no exogenous proteins for 2 h; 2) no exogenous proteins for 1 h and 200 nM proMmp-9 or proMmp-8 alone for 1 h; 3) 200 nM Timp-1 for 1 h and then 200 nM proMmp-9 or proMmp-8 for 1 h; 4) 200 nM Timp-2 for 1 h and then 200 nM proMmp-9 or proMmp-8 for 1 h; The cells were fixed and then incubated at 37°C with: 1) gelatin conjugated to quenched FITC for 18 h (in E); or 2) type-I collagen conjugated to quenched FITC for 18 h (in F). In E-F, Mmp-mediated cleavage of each substrate in the presence of amino-phenyl mercuric acetate was quantified in cell-free supernatant samples using fluorimetry, as described in Methods. The data are expressed as mean + SEM % cleavage of the substrate in cells incubated without exogenous proteins; n=4–5 separate experiments. Data were analyzed using a Kruskal-Wallis One-Way ANOVA followed by pair-wise testing with Mann-Whitney U tests. Asterisks indicate P < 0.05 compared with all other groups.

Techniques: Incubation, Two Tailed Test, MANN-WHITNEY

In A, PAF- and fMLP-activated Mmp-8−/− x Mmp-9−/− PMNs were incubated for 2 h at 4°C with or without 400 nM full length proMmp-8 protein (FL Mmp8) or 400 nM mutant proMmp-8 protein lacking the COOH-terminal hemopexin domain (MT Mmp-8). Bound Mmp-8 was detected by immunostaining cells with Alexa 488 using an antibody raised to the hinge region of Mmp-8 (which is present in both the FL and MT forms of Mmp-8). In B, PAF- and fMLP-activated Mmp-8−/− x Mmp9−/− PMNs were incubated for 2 h at 4°C with or without 400 nM full length Mmp-9 (FL Mmp9) or 400 nM mutant Mmp-9 lacking the COOH-terminal hemopexin domain (MT Mmp9). Bound Mmp-9 was detected by immunostaining cells with Alexa 488 using an antibody raised to the hinge region of Mmp-9 (which is present in both the FL and MT forms of Mmp-8 and Mmp-9). In A-B, Data are mean + SEM; n = 3 separate experiments each analyzing 300 cells/group). Data were analyzed using a One-Way ANOVA followed by pair-wise testing with two-tailed Student’s t-tests. Asterisk indicates P < 0.05 compared with the no exogenous Mmp group or the group indicated. In C-D, PAF- and fMLP-activated Mmp-8−/− x Mmp9−/− PMNs were incubated at 4°C for 60 min with or without 500 nM soluble murine hemopexin protein. Cells were then incubated at 4°C for 60 min with or without 400 nM exogenous proMmp-8 or 400 nM exogenous proMmp-9. Mmp-8 and Mmp-9 that bound to cells was detected by immunostaining the cells with Alexa-488 for Mmp-8 (C) or Mmp-9 (D), as described above. Data are mean + SEM; n = 3 separate experiments each analyzing 300 cells/group. Data were analyzed using a One-Way ANOVA followed by pair-wise testing with two-tailed Student’s t-tests. Asterisk indicates P < 0.05 compared with the group incubated without exogenous proteins or the group indicated.

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Tissue Inhibitor of Metalloproteinase-1 (TIMP-1) Promotes PMN Pericellular Proteolysis by Anchoring Matrix Metalloproteinase-8 and -9 to PMN Surfaces

doi: 10.4049/jimmunol.1801466

Figure Lengend Snippet: In A, PAF- and fMLP-activated Mmp-8−/− x Mmp-9−/− PMNs were incubated for 2 h at 4°C with or without 400 nM full length proMmp-8 protein (FL Mmp8) or 400 nM mutant proMmp-8 protein lacking the COOH-terminal hemopexin domain (MT Mmp-8). Bound Mmp-8 was detected by immunostaining cells with Alexa 488 using an antibody raised to the hinge region of Mmp-8 (which is present in both the FL and MT forms of Mmp-8). In B, PAF- and fMLP-activated Mmp-8−/− x Mmp9−/− PMNs were incubated for 2 h at 4°C with or without 400 nM full length Mmp-9 (FL Mmp9) or 400 nM mutant Mmp-9 lacking the COOH-terminal hemopexin domain (MT Mmp9). Bound Mmp-9 was detected by immunostaining cells with Alexa 488 using an antibody raised to the hinge region of Mmp-9 (which is present in both the FL and MT forms of Mmp-8 and Mmp-9). In A-B, Data are mean + SEM; n = 3 separate experiments each analyzing 300 cells/group). Data were analyzed using a One-Way ANOVA followed by pair-wise testing with two-tailed Student’s t-tests. Asterisk indicates P < 0.05 compared with the no exogenous Mmp group or the group indicated. In C-D, PAF- and fMLP-activated Mmp-8−/− x Mmp9−/− PMNs were incubated at 4°C for 60 min with or without 500 nM soluble murine hemopexin protein. Cells were then incubated at 4°C for 60 min with or without 400 nM exogenous proMmp-8 or 400 nM exogenous proMmp-9. Mmp-8 and Mmp-9 that bound to cells was detected by immunostaining the cells with Alexa-488 for Mmp-8 (C) or Mmp-9 (D), as described above. Data are mean + SEM; n = 3 separate experiments each analyzing 300 cells/group. Data were analyzed using a One-Way ANOVA followed by pair-wise testing with two-tailed Student’s t-tests. Asterisk indicates P < 0.05 compared with the group incubated without exogenous proteins or the group indicated.

Techniques: Incubation, Mutagenesis, Immunostaining, Two Tailed Test

In A and B, human PMNs were incubated with 1 μg/mL LPS for 4 h at 37°C to induce NET formation, or without LPS as a control. In A, the cells were fixed and then double immunostained with a green fluorophore for TIMP-1 (second panels) and a red fluorophore for MMP-8 or MMP-9 (third panels). Nuclei were counterstained blue with 4’,6-diamidino-2-phenylindole (DAPI). Co-localization of TIMP-1 and MMPs in PMN-associated NETs was assessed by confocal microscopy (see merged images in the fourth panels). The results shown are representative of 4 different PMN preparations. In B, NET induction by LPS was quantified by staining the samples for extracellular DNA with SYTOX™ Blue Nucleic Acid Stain, and quantifying the staining as described in Methods. Data are mean + SEM; n = 4 experiments. Data were analyzed using the Mann-Whitney U tests. Asterisk indicates P < 0.05 compared with unstimulated cells.

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Tissue Inhibitor of Metalloproteinase-1 (TIMP-1) Promotes PMN Pericellular Proteolysis by Anchoring Matrix Metalloproteinase-8 and -9 to PMN Surfaces

doi: 10.4049/jimmunol.1801466

Figure Lengend Snippet: In A and B, human PMNs were incubated with 1 μg/mL LPS for 4 h at 37°C to induce NET formation, or without LPS as a control. In A, the cells were fixed and then double immunostained with a green fluorophore for TIMP-1 (second panels) and a red fluorophore for MMP-8 or MMP-9 (third panels). Nuclei were counterstained blue with 4’,6-diamidino-2-phenylindole (DAPI). Co-localization of TIMP-1 and MMPs in PMN-associated NETs was assessed by confocal microscopy (see merged images in the fourth panels). The results shown are representative of 4 different PMN preparations. In B, NET induction by LPS was quantified by staining the samples for extracellular DNA with SYTOX™ Blue Nucleic Acid Stain, and quantifying the staining as described in Methods. Data are mean + SEM; n = 4 experiments. Data were analyzed using the Mann-Whitney U tests. Asterisk indicates P < 0.05 compared with unstimulated cells.

Techniques: Incubation, Confocal Microscopy, Staining, MANN-WHITNEY

NHPs were exposed to air (n = 4; white bars) or smoke (n = 6; gray bars) for 4 weeks, and PMNs were isolated from peripheral blood samples from each animal both at baseline and after 4 weeks of air or CS exposure. Cells were double immunostained with Alexa 488 and murine anti-TIMP-1 IgG or non-immune murine (Ms) IgG (A and B second panels) and with Alexa 546 and rabbit anti-MMP-8 IgG or rabbit anti-MMP-9 IgG (A and B third panels). Nuclei in the cells were counterstained blue using 4’,6-diamidino-2-phenylindole (DAPI, A and B first panels). Co-localization of TIMP-1 and MMPs on the surface of PMNs was assessed using confocal microscopy (see merged images in the fourth panels in A and B). The white bars are 5 microns in length. In C-E, surface TIMP-1 or MMPs levels were quantified using image analysis software, as described in Methods. Data are mean + SEM; n = 4 air-exposed NHPs and n = 6 smoke-exposed NHPs (200–500 cells were analyzed per animal). In C-E, data were analyzed using a One-Way ANOVA followed by pair-wise testing with two-tailed Student’s t-tests. Asterisk indicates P < 0.001 compared with baseline or the group indicated.

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Tissue Inhibitor of Metalloproteinase-1 (TIMP-1) Promotes PMN Pericellular Proteolysis by Anchoring Matrix Metalloproteinase-8 and -9 to PMN Surfaces

doi: 10.4049/jimmunol.1801466

Figure Lengend Snippet: NHPs were exposed to air (n = 4; white bars) or smoke (n = 6; gray bars) for 4 weeks, and PMNs were isolated from peripheral blood samples from each animal both at baseline and after 4 weeks of air or CS exposure. Cells were double immunostained with Alexa 488 and murine anti-TIMP-1 IgG or non-immune murine (Ms) IgG (A and B second panels) and with Alexa 546 and rabbit anti-MMP-8 IgG or rabbit anti-MMP-9 IgG (A and B third panels). Nuclei in the cells were counterstained blue using 4’,6-diamidino-2-phenylindole (DAPI, A and B first panels). Co-localization of TIMP-1 and MMPs on the surface of PMNs was assessed using confocal microscopy (see merged images in the fourth panels in A and B). The white bars are 5 microns in length. In C-E, surface TIMP-1 or MMPs levels were quantified using image analysis software, as described in Methods. Data are mean + SEM; n = 4 air-exposed NHPs and n = 6 smoke-exposed NHPs (200–500 cells were analyzed per animal). In C-E, data were analyzed using a One-Way ANOVA followed by pair-wise testing with two-tailed Student’s t-tests. Asterisk indicates P < 0.001 compared with baseline or the group indicated.

Techniques: Isolation, Confocal Microscopy, Software, Two Tailed Test

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polyclonal rabbit anti murine mmp 9 igg (Bio-Rad)

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