In vivo PLD activity was determined by PLD-catalyzed transphosphatidylation as previously described20 (link). Briefly, 3T3-L1 cells were metabolically labeled with 3H-oleic acid for 18 h. After pretreatment with 0.3% 1-butanol for 30 min, cells were lysed and lipids were extracted according to the method of Bligh and Dyer57 (link) and analyzed by TLC. Alternatively, a Phospholipase D Assay Kit (Sigma Aldrich) was also used, as indicated in the figure legends, to measure PLD activity according to the manufacturer’s instructions58 (link)59 (link)60 (link)61 (link). To calculate recombinant PLD1 or PLD2 activity specifically, the PLD activity in cells transfected with empty vector was subtracted from the activity in PLD1 or PLD2-transfected cells under the same conditions20 (link)62 (link). Cells were transfected with HA-PLD120 (link) and HA-PLD220 (link) using Amaxa Nucleofector technology (Amaxa, Cologne, Germany) according to manufacturers’ protocol.
Phospholipase d assay kit
Manufactured by Merck Group
Sourced in United States
The Phospholipase D Assay Kit is a laboratory tool used to measure the activity of the enzyme phospholipase D in biological samples. The kit provides reagents and protocols for the colorimetric detection and quantification of phospholipase D.
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5 protocols using phospholipase d assay kit
1
Measuring Phospholipase D Activity in Cells
2
Phospholipase D Activity Colorimetric Assay
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Phospholipase D Assay Kit was used following the manufacture’s recommended protocol (Sigma, Saint Louis, Missouri, USA, Cat. No.MAK137). In this assay, PLD hydrolyzes phosphatidylcholine to choline, which is determined using choline oxidase resulting in a colorimetric (570 nm) /fluorometric(λex=530/λem=585 nm) product, proportional to the PLD activity in the sample. Three technical and three biological replicates were used in this assay, initial and final absorbance were measured at 570 nm using Epoch 2 Microplate (Biotek, Nepean, Ontario, Canada). The slope of the calibrator curve was used to calculate the phospholipase D activity of the sample using the following equation:
where t=enzyme reaction time (20 min in standard assay) and n=dilution factor. Unit definition: one unit of PLD catalyzes the formation of 1 mmole choline per min under the assay conditions (pH 7.4).
where t=enzyme reaction time (20 min in standard assay) and n=dilution factor. Unit definition: one unit of PLD catalyzes the formation of 1 mmole choline per min under the assay conditions (pH 7.4).
3
Phospholipase D Assay Protocol
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A phospholipase D assay kit (Sigma Aldrich, St. Louis, MO, USA) was used according to the manufacturer’s instructions.
4
Autotaxin PLD Activity Assay
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Autotaxin belongs to the ENPP family of enzymes and displays phospholipase D (PLD) activity. PLD activity was measured using phospholipase D assay kit (Sigma, Missouri, USA), according to manufacturer’s instructions. Briefly, BV-2 cells were seeded onto 6-well plates at a density of 1 × 105 cells/well and serum-starved overnight prior to experiment. Then, cells were treated with LPS (20 ng/mL) in absence or presence of the indicated concentrations of PF8380 for the indicated time periods. DMSO was used as vehicle control. At the end of the time points, the medium was collected. Then, 10 µL of the supernatant was mixed with 90 µL of the master reaction. After 10 min, initial absorbance, (A570) initial, was measured at 570 nm. The plate was incubated at room temperature for 20 min and absorbance was measured again to determine (A570) final. One unit of PLD catalyzes the formation of 1 µmole choline per minute under the assay condition, and is calculated as follows:
Sample PLD activity = (B/(∆T × V)) × D
where the following applies:
B = amount of choline in the sample (nmol);
∆T = reaction time (minutes);
V = sample volume added into the reaction well (mL);
D = sample dilution factor.
Sample PLD activity = (B/(∆T × V)) × D
where the following applies:
B = amount of choline in the sample (nmol);
∆T = reaction time (minutes);
V = sample volume added into the reaction well (mL);
D = sample dilution factor.
5
Liver Lysosome Isolation and PLD Activity
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Lysosomal fractions were isolated from the liver using a Lysosome Isolation Kit (LYSISO1, Sigma Aldrich) according to the manufacturer instructions. PLD activity was measured using Phospholipase D Assay Kit (MAK137, Sigma Aldrich) according to the manufacturer instructions.
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