Extracellular cathepsin activity was assessed as previously described by our laboratory (Sager et al., 2014 (link)). Briefly, 2 µg Z-LR-AMC (specific to cathepsin B, cathepsin L and cathepsin V; R&D systems) in PBS was added to 50 µL of whole lung lavage fluid in a total reaction volume of 150 µL. The assays were incubated at 37°C for 1 h then fluorescence was measured using a plate reader at 380 nm excitation and 460 nm emission.
Z lr amc
Manufactured by R&D Systems
Sourced in United States
Z-LR-AMC is a laboratory equipment product manufactured by R&D Systems. It is designed to perform a specific function, but I do not have enough detailed and unbiased information to provide a concise description of its core function without the risk of extrapolation or interpretation. Therefore, I cannot provide a detailed description while maintaining an unbiased and factual approach.
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Lab products found in correlation
5 protocols using z lr amc
1
Extracellular Cathepsin Activity Assay
2
Cathepsin Activity Fluorometric Assay
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Fluorometric determination of cathepsins B, S and L activities was achieved by incubation of recombinant human cathepsins B, S, and L (Novoprotein, Summit, NJ, USA) with their respective specific fluorogenic substrates (Cat B: ZLR-AMC (R&D Systems, Minneapolis, MN, USA)); Cat L/S: Ac-HRYR-ACC (Calbiochem, Etobicoke, ON, Canada)) and increasing concentrations of LHVS (0.1563, 0.3125, 0.625, 1.25, 2.5, 5 ug/ml) in an assay buffer (20 mM sodium acetate pH 5.5 containing 0.675 mM KCl, 0.25 mM CaCl2, 0.125 mM MgCl2) supplemented with 500 μM L-cysteine:cystine (600:1 molar ratio) at 37°C in a FLUOstar Optima fluorescent plate reader (BMG Labtech, Ortenberg, Germany) as previously described [34 (link), 40 (link)]. Relative hydrolytic activities were determined by calculating the slopes of the increasing substrate fluorescence (y = mx+c; where y indicates relative fluorescence, m indicates slope, x indicates time) of the initial reaction (20 min) and expressed relative to uninhibited samples.
3
Measurement of Inflammatory Mediators in Lung Samples
4
Cathepsin Activity Determination in Plasma
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Cathepsin activity for the blood plasma was determined by mixing the following assay components in a 96-well plate using PBS as diluent: first blood plasma (50 μl), 2 μg Z-LR-AMC (fluorogenic peptide substrate, R&D systems, Minneapolis, MN) in a total volume of 150 μl. The assays samples were incubated at 37 °C for 1 h, then fluorescence was measured using a plate reader at 380 nm excitation and 460 nm emission.
5
Fluorogenic Assay for Cathepsin-B Activity
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As previously described by our laboratory [23 (link)], to determine total and B-specific cathepsin activities the following assay components were mixed in a 96-well plate using PBS as diluent: first WLL fluid (50 μL), 2 μg Z-LR-AMC (fluorogenic Peptide Substrate, R & D systems, Minneapolis, MN, USA) ± 66 μM inhibitor (Z-Phe-Phe-FMK, MBL International, Woburn, MA, USA) in a total volume of 150 μL. The assays samples were incubated at 37°C for 1 h then fluorescence was measured using a plate reader at 380 nm excitation and 460 nm emission. Cathepsin-B specific activity was calculated as follows: relative fluorescence units (RFU) from assay without inhibitor minus the assay with inhibitor.
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