MMP activity (MMP2 and MMP9) was measured by a fluorogenic peptide substrate (R&D Systems), according to the protocol recommended by the manufacturer. Briefly, the MMP substrate was diluted in TCN buffer (150 mmol/L NaCl, 10 mmol/L CaCl2, 50 mmol/L Tris-HCl; pH 7.5), and then was added to the supernatants (preactivated by aminophenylmercuric acetate for 1 hour) before incubation at 37 °C. After 30 minutes, total MMP activity was determined on a fluorometer (FLX 800 Microplate Fluorescence Reader; Bio-Tek Instruments, Winooski, VT).
Fluorogenic peptide substrate
Manufactured by R&D Systems
Sourced in United States
The Fluorogenic peptide substrate is a laboratory tool used to measure the activity of enzymes that cleave peptide bonds. It consists of a short peptide sequence labeled with a fluorescent dye. When the enzyme cleaves the peptide, the dye is released, resulting in a measurable increase in fluorescence. This product can be used to quantify the enzymatic activity of proteases and other peptidases in research applications.
Automatically generated - may contain errors
Lab products found in correlation
Flx800 fluorescence microplate reader, by Agilent Technologies (3 mentions)
Ethylenediaminetetraacetic acid tubes, by Sarstedt (1 mentions)
Procarta multiplex cytokine kit, by Thermo Fisher Scientific (1 mentions)
Advia 2120i, by Siemens (1 mentions)
Microplate fluorometer, by Thermo Fisher Scientific (1 mentions)
Ascent software v2, by Thermo Fisher Scientific (1 mentions)
Milliq 50 glycerol, by Merck Group (1 mentions)
Fluoroskan ascent, by Thermo Fisher Scientific (1 mentions)
6 protocols using fluorogenic peptide substrate
1
Quantifying MMP2 and MMP9 Activity
2
Quantitative Analysis of MMP-2 and MMP-9
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MMP-2 and MMP-9 activity was measured using a fluorogenic peptide substrate (R&D Systems) following the manufacturer's protocol. Briefly, the MMPs substrate was diluted in TCN buffer (50 mmol/L Tris-HCl, 150 mmol/L NaCl and 10 mmol/L CaCl2; pH 7.5) and added to supernatants from corneal or cell lysates (pre-activated by aminophenylmercuric acetate for 1 hour) before incubation at 37°C. After 30 minutes, total MMP activity was determined on a fluorometer (FLX 800 Microplate Fluorescence Reader; Bio-Tek Instruments, Winooski, VT).
3
Hematology Analysis and Biomarker Measurement
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At each timepoint, 20 ml of peripheral blood was collected in lithium heparin tubes, and blood cell counts were measured using a hematology analyzer (ADVIA 2120i, Siemens, Zurich, Switzerland) (Kim et al., 2012 (link)). White blood cell counts were read from the peroxidase channel. If necessary, samples were diluted (1:3, 1:8). Platelet counts are not displayed in the results section, since lithium-heparin anticoagulated blood is more prone to platelet clumping and might bias outcome (Nelles and Chandler, 2014 (link)).
Additional blood was collected in 9 ml serum and ethylenediaminetetraacetic acid tubes (Sarstedt, Nümbrecht, Germany). Tubes were centrifuged according to the manufacturer’s instructions and immediately frozen (−80°C) until analysis. Parameters of endothelial matrix remodeling (MMP-2 and -9; TIMP-1) were determined by ELISAs (R&D Systems Europe, United Kingdom). MMP activity was analyzed using Fluorogenic Peptide Substrate (R&D Systems Europe, United Kingdom) (Bernecker et al., 2011 (link)). Parameters of hematopoietic stem cell mobilization (SDF-1α; IL-3) were measured by Procarta multiplex cytokine kit (Affymetrix, Santa Clara, CA, United States) (Schafer et al., 2016 (link)). Inter- and intra-assay CVs for ELISAs were below 15%.
Additional blood was collected in 9 ml serum and ethylenediaminetetraacetic acid tubes (Sarstedt, Nümbrecht, Germany). Tubes were centrifuged according to the manufacturer’s instructions and immediately frozen (−80°C) until analysis. Parameters of endothelial matrix remodeling (MMP-2 and -9; TIMP-1) were determined by ELISAs (R&D Systems Europe, United Kingdom). MMP activity was analyzed using Fluorogenic Peptide Substrate (R&D Systems Europe, United Kingdom) (Bernecker et al., 2011 (link)). Parameters of hematopoietic stem cell mobilization (SDF-1α; IL-3) were measured by Procarta multiplex cytokine kit (Affymetrix, Santa Clara, CA, United States) (Schafer et al., 2016 (link)). Inter- and intra-assay CVs for ELISAs were below 15%.
4
Enzymatic Activity of Snake Venom
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A working stock solution of freeze dried venom was reconstituted in a buffer containing 50% MilliQ/50% glycerol (>99.9%, Sigma, St. Louis, MO, USA) at a 1:1 ratio to preserve enzymatic activity and reduce enzyme degradation. Varying concentrations of crude venom (10 ng/µL and 50 ng/µL) were plated out in triplicates on a 384-well plate (black, Lot#1171125, nunc™ Thermo Scientific, Rochester, NY, USA) and measured by adding 90 µL quenched fluorescent substrate per well (total volume 100 µL/well, 10 µL/5mL enzyme buffer, 150 mM NaCl, and 50 mM Tri-HCl (pH 7.3), Fluorogenic Peptide Substrate, R&D systems, Cat#ES001 & ES011, Minneapolis, MI, USA). Fluorescence was monitored by a Fluoroskan Ascent™ (Thermo Scientific, Vantaa, Finland) Microplate Fluorometer (Cat#1506450, Thermo Scientific, Vantaa, Finland) (Cat#ES001 for Matrix Metalloprotease at an excitation of 320 nm, emission at 405 nm; Cat#ES011 for Kallikrein at an excitation of 390 nm, emission at 460 nm) over 400 mins or until activity had ceased. Data was collected using Ascent® Software v2.6 (Thermo Scientific, Vantaa, Finland).
5
Enzymatic Activity Assay of Venom
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A working stock solution of freeze dried venom was reconstituted in a buffer containing 50% deionised H2O/50% glycerol (>99.9%, Sigma-Alrich) at a 1:1 ratio to preserve enzymatic activity and reduce enzyme degradation with the final venom concentration of 0.1 mg/mL, and then stored at −20 °C. Venom solutions (1 µg in dry venom weight) were plated in triplicates on a 384 well plate and measured by adding 90 µL quenched fluorescent substrate per well (total volume 100 µL/well; 10 µL/5mL enzyme buffer—150 mM NaCl and 50 mM Tri-HCl pH 7.4, Fluorogenic Peptide Substrate, R & D systems Cat#ES0011, Minneapolis, MN, USA). Fluorescence was monitored (excitation at 390 nm and emission at 460 nm) over 400 min or until activity ceased.
6
Fluorogenic Assay for MMP-2 and MMP-9
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MMP-2 and MMP-9 activity in corneas or RAW264.7 cells was measured using a fluorogenic peptide substrate (R&D Systems) according to the manufacturer’s protocol. In brief, the MMPs substrate was diluted in TCN buffer (50 mmol/L Tris-HCl, 10 mmol/L CaCl2 and 150 mmol/L NaCl; pH 7.5) and added to supernatants from corneal or RAW264.7 cell lysates (pre-activated by aminophenylmercuric acetate for 1 hour). 30 minutes later, total MMP activity was determined with FLX 800 Microplate Fluorescence Reader (Bio-Tek Instruments, Winooski, VT).
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