PLC activity assay was performed using total protein lysate of human articular chondrocytes after 3 days of lentiviral IGF-II and lentiviral GFP infection. Two types of PLC activities were assessed. The PC-PLC activity, which releases inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol (DAG), was determined using the EnzChek direct phospholipase C assay kit (Invitrogen). The PI-PLC activity, which releases phosphocholine and DAG, was assayed using an established protocol [Burg-Golani et al., 2013 (link)]. Briefly, 1 μg of total protein lysate diluted in 100 μl water was mixed with 100 μl of assay solution (0.036 g phosphatidyl-inositol in 1 ml of 58 mM sodium-cholate, 10 mM CaCl2, and 7 ml of 0.15M NaCl) and incubated at 37°C overnight. Then the photo-absorbance at 510 nm was measured as the relative PI-PLC activity. Three independent experiments were performed in triplicate.
Enzchek direct phospholipase c assay kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The EnzChek direct phospholipase C assay kit is a fluorescence-based assay designed to detect and quantify phospholipase C (PLC) activity. It provides a direct method for measuring PLC activity by monitoring the release of a fluorescent dye from a phospholipid substrate.
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4 protocols using enzchek direct phospholipase c assay kit
1
Quantifying PLC Activity in Chondrocytes
2
Phospholipase C Activity Measurement
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ALL cells were treated with or without test compounds alone or in combination for 24 h. The measurement of phospholipase C activity was assessed using the EnzChek™ Direct Phospholipase C Assay Kit (Invitrogen, no: E10215) according to the manufacturer’s instructions.
3
Measuring Phospholipase C Activity
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10.000.000 BMDMs were plated in petri dishes overnight and primed with LPS as described above. The cells were then pre-incubated with arachidonic acid or U-73122 as described above and next stimulated with nigericin 10 μM for 1 h with or without inhibitors. The cells were then collected and lysed for 10 min in ice using buffer containing 10 mM Tris pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 10 mM NaF, 1 mM NaVO4, 20 mM PMSF, Phosphatase inhibitor cocktail 3 (1 in 100 dilution, Sigma) and Protease inhibitor cocktail (1 in 100 dilution, P8340, Sigma). PLC activity was then measured with EnzChek Direct Phospholipase C Assay Kit (Thermo) as indicated by the manufacturer.
4
Lipase and Phospholipase Activity Assays
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Lipase and phospholipase assays using the purified human EPDR1 were performed according to the manufacturers’ protocols. EPDR1 did not show any activities using the Lipase Activity Assay Kit (catalog No. MAK046; Sigma–Aldrich, St Louis, Missouri, USA), the Phospholipase D Activity Assay Kit (catalog No. MAK137; Sigma–Aldrich) or the EnzChek Direct Phospholipase C Assay Kit (catalog No. E10215; Thermo Fisher Scientific, Waltham, Massachusetts, USA).
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