Gelatin zymography assay was carried out as described previously with some modifications (Arai, Lee, & Lo, 2003 (link); Seo et al., 2013 (link); Y. Takahashi et al., 2014 (link)). In brief, conditioned media (CM) from microglial cultures, which were treated with baits or LPS (Sigma), were collected, concentrated 20x by using VIVASPIN 500 (10,000 MWCO PES, sartorius), and mixed with an equal volume of 2x Tris-Glycine SDS Sample Buffer (novex). Following electrophoresis on Novex 10% Zymogram Plus (Gelatin) Gel (Invitrogen), gels were incubated with Renaturing Buffer (Invitrogen) at RT for 30 min, Developing Buffer (Invitrogen) at RT for 30 min, and fresh Developing Buffer at 37C for 24 h. Gels were then stained with staining solution (42% (v/v) methanol, 8% (v/v) acetate acid, 0.5%(w/v) coomasie brilliant blue) at RT for 2 h, and de-stained with de-staining solution (40% (v/v) methanol, 10% (v/v) acetate acid) at RT for 30 min. Densitometry of bands on the gels was calculated by Image J by operators who did not know the group distribution. LPS (Sigma)-activated microglia were prepared by incubation with 1 μg/μl of LPS at 37°C for 2h with or without inhibitors, and then, maintained in the DMEM/F12 containing 1% P/S at 37°C for 24 h.
Developing buffer
Manufactured by Thermo Fisher Scientific
Sourced in United States
Developing buffer is a solution used to facilitate the development of biological samples in various laboratory applications. It serves as a medium to maintain the appropriate pH and ionic conditions necessary for optimal sample processing. The core function of the developing buffer is to provide a controlled environment that supports the intended analysis or experimental procedures.
Automatically generated - may contain errors
Lab products found in correlation
Renaturing buffer, by Thermo Fisher Scientific (35 mentions)
Simplyblue safestain, by Thermo Fisher Scientific (21 mentions)
Precast zymography gels, by Thermo Fisher Scientific (6 mentions)
Gelatin, by Merck Group (5 mentions)
Renaturation buffer, by Thermo Fisher Scientific (4 mentions)
Renaturating buffer, by Thermo Fisher Scientific (4 mentions)
Zymogram renaturing buffer, by Thermo Fisher Scientific (3 mentions)
Coomassie blue r 250, by Bio-Rad (3 mentions)
Novex 10 zymogram gelatin gel, by Thermo Fisher Scientific (3 mentions)
Lipopolysaccharides (lps), by Merck Group (2 mentions)
Zymogram gelatin gel, by Thermo Fisher Scientific (2 mentions)
Coomassie brilliant blue r 250, by Bio-Rad (2 mentions)
Novex zymogram gel, by Thermo Fisher Scientific (2 mentions)
Zymography gels, by Bio-Rad (2 mentions)
Imagelab software version 4, by Bio-Rad (2 mentions)
Novex zymogram gelatin gel, by Thermo Fisher Scientific (2 mentions)
Tris glycine sds running buffer, by Thermo Fisher Scientific (2 mentions)
Tris glycine sds native sample buffer, by Thermo Fisher Scientific (2 mentions)
Coomassie blue, by Merck Group (2 mentions)
Azure c500, by Azure Biosystems (2 mentions)
60 protocols using developing buffer
1
Gelatin Zymography for Microglial Matrix Metalloproteinases
2
Gelatin Zymography of Conditioned Media
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Gelatin zymography of conditioned media was performed as described previously [34 (link)]. Briefly, conditioned media from the different treatment conditions were diluted 1:1 in nonreducing sample buffer and separated on 10% SDS polyacrylamide gels containing 0.1% gelatin (Thermo-Fisher Scientific, Waltham, MA USA) for 150 min at 125 V. SDS was removed by incubation with renaturing buffer (2.5% Triton X-100 diluted in water) for 30 min at room temperature. The gels were washed for 30 min in developing buffer (Thermo-Fisher Scientific, Waltham, MA USA) and then incubated overnight at 37 °C in fresh developing buffer. Finally, the gels were stained with Coomassie blue. Zones of enzymatic (gelatinolytic) activity were characterized by the absence of Coomassie blue staining.
3
Gelatin Zymography of Micronized dHACM
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Micronized dHACM (n = 4) was incubated in Tris glycine SDS (ThermoFisher) overnight at 4°C with gentle agitation at a concentration of 40 mg/mL. Samples were centrifuged and filtered before dilution to concentrations of 20, 10, and 1 mg/mL for each donor. Ten nanograms of recombinant MMP-2 (R&D Systems) that had been activated for 1 h at 37°C with 4-aminophenylmercuric acetate (APMA) was used as a control, and a Benchmark Pre-stained protein ladder (Novex) was included with each gel. Each sample was loaded into individual wells of a 10% gelatin zymogram gel (Novex) and run at 125 V for 110 min. Gels were collected and incubated in 1 × Renaturing buffer (Novex) for 30 min, followed by 1 × Developing buffer (Novex) overnight at 37°C. Gels were stained with SimplyBlue™ SafeStain (ThermoFisher) for 30 min and destained in 20% NaCl for 24 h. Images were taken by using an Amersham Imager 600 (GE Healthcare Life Sciences) and analyzed by using ImageQuantTL software. Band densities were quantified and normalized to the recombinant MMP-2 control.
4
Gelatin Zymography for MMP2 and MMP9
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MMP2 and MMP9 activations were examined in supernatants harvested from uninfected or P. gingivalis-infected OECs culture using gelatin zymography. Protein samples were precipitated from the cell culture supernatant using TCA precipitation method (Link and LaBaer, 2011 (link)) and loaded on 10% SDS-polyacrylamide gel containing 3% gelatin. The gels were incubated at ambient temperature with renaturing buffer (Novex) two times for 30 min each, followed by overnight incubation at 37°C with developing buffer (Novex). After staining with Coomassie Brilliant Blue R-250, enzymatic activities of MMP2 and MMP9 were visualized as clear bands against a dark blue background.
5
Procyanidin Inhibits TLR4-Mediated Signaling
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Procyanidin was purchased from Aladdin Co. Ltd. (Shanghai, China). Secondary antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Toll-like receptor 4 (TLR-4) inhibitor TAK-242, phosphatidylinositol 3-kinase (PI3K) inhibitor AS-605240, and protein kinase B (Akt) inhibitor GSK690693 were purchased from Selleck Chemicals (Shanghai, China). RAGE antibody 553030 was purchased from Calbiochem (Merck, Darmstadt, Germany). Anti-HMGB1 polyclonal antibody 326052233 was purchased from SHINO-TEST Corporation (Sagamihara City, Japan) and GAPDH was purchased from Proteintech, Wuhan, China. Gelatin was purchased from Amresco (Solon, OH, USA). Zymogram renaturing buffer and developing buffer were purchased from Novex (Carlsbad, CA, USA). FBS was purchased from Gibco (Grand Island, NY, USA), and other cell culture media and supplements were purchased from HyClone (Logan, UT, USA). All other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA).
6
Gelatinase Activation Assay for Exosomes
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Equal amounts of exosomes/latex complex were resuspended in 20 μL detergent free-MMP activation buffer (50 mM Tris-HCl, 50 mM NaCl, 10 mM CaCl2, 1 μM ZnSO4, pH 7.5) with a total of 50 ng of pro-MMP2 (Calbiochem). Samples were incubated at 37°C for 4 hours and then mixed with 5 μL of reducing agent free-4X SDS loading dye (Invitrogen). Samples were loaded and separated on a 10% zymogram gelatin gel (Invitrogen). The zymogram gel was rinsed with deionized water and then incubated with denaturing buffer (Invitrogen) for 2 hours to remove the SDS from the gel. Each gel was incubated overnight with developing buffer (Invitrogen). Coomassie brilliant blue R-250 (Bio-Rad) was used to stain the gel, and images were taken using the ChemiDOC image system (Bio-Rad). Pro-MMP2 with only aldehyde/sulfate latex was used as a control.
7
Quantifying Active MMP-9 via Gelatin Zymography
8
Gelatin Zymography of Conditioned Media
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gelatin zymography of conditioned media was performed as described previously [28 (link)]. Briefly, conditioned media from the different treatment conditions were diluted 1:1 in non-reducing sample buffer and separated on 10 % SDS polyacrylamide gels containing 0.1 % gelatin (Invitrogen) for 150 min at 125 V. SDS was removed by incubation with renaturing buffer (Triton X-100, 2.5 % diluted in water) for 30 min at room temperature. The gels were washed for 30 min in developing buffer (Invitrogen) and then incubated overnight at 37 °C in fresh developing buffer. Finally, gels were stained with Coomassie blue. Zones of enzymatic (gelatinolytic) activity were characterized by the absence of Coomassie blue.
9
Gelatin Zymography for MMP-2 Activity
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The activity of MMP-2 was evaluated using zymography. Zymography was performed using 10% polyacrylamide gels in the presence of gelatin (0.5 mg/mL) as a substrate for MMP-2. The samples were suspended in loading buffer (10% SDS, 25% glycerol, 0.25 M Tris (pH 6.8) and 0.1% bromophenol blue) and loaded onto 10% SDS-PAGE gels without denaturation. After electrophoresis, the gels were incubated in renaturing buffer (Invitrogen) at room temperature for 30 min and then incubated for 24 h at 37 °C in developing buffer (Invitrogen). The gels were then stained with 0.5% Coomassie Brilliant Blue.
10
Gelatin Zymography for MMP-9 Activity
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