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Protocol
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1. Sample Preparation: a. For microplate cultured 3T3 cells: Culture cells in a 96-well plate, treat cells with desired reagents and methods. When the cells are ready for triglyceride testing, remove medium completely from wells and wash once with PBS. Add 100 µl Lipid Extraction Solution per well, seal plate with an adhesive cover to prevent evaporation. Place entire plate* in plate heater or heating block at 90-100°C for 30 min. Solution in the wells will become cloudy when heated. Cool plate to room temperature. Mix solution by shaking plate for 1 minute. Triglycerides are now completely dissolved in the Lipid Extraction Buffer. b. For tissue (0.1-10 mg) or cells (1,000-1 million): Homogenize samples in 100 μl Lipid Extraction Solution then heat/vortex as described above. Centrifuge briefly (top speed using a bench top centrifuge) to remove debris/insoluble material. If oil droplets are still observed, reduce the number of cells used per assay. In our hands, ~1,000-10,000 differentiated 3T3 cells using 100 μl Lipid Extraction Solution are sufficient for the colorimetric assay. For triglyceride assay, transfer 5-50μl** of the lipid extracts to 96-well plate, bring the volume to total 50 μl with Assay Buffer. Notes: *If only a few wells are to be tested, not the whole plate, pipette the Extraction Solution up and down 3-4 times in the culture wells, rinsing the well bottom to fully suspend the lipid droplets in the Lipid Extraction Solution. Complete mixing can be confirmed under microscope under 4-10X power, droplets will be seen uniformly dispersed through the depth of the Extraction Solution, not associated with the well bottom. **3T3 cells can accumulate exceedingly large amounts of triglyceride. Fully differentiated cells can contain 100X the amount of triglyceride as uninduced cells. The amount of the lipid extract used for the triglyceride assay will depend on cell type, treatment and cell differentiation stage. For unknown samples, we suggest testing different doses of your sample to make sure the readings are within the standard curve range. **Protein concentration of the lipid extracts can be tested and used as an internal control to normalize the lipid concentration in the sample. We suggest using a detergent insensitive protein assay such as the BCA method for the protein assay. 2. Standard Curve Preparation: For the colorimetric assay, dilute 40 µl of the 1 mM Triglyceride standard into 160 µl Assay Buffer, mix to generate 0.2 mM standard. Add 0, 10, 20, 30, 40, 50 µl of the 0.2 mM Triglyceride Standard into a series of wells. Adjust volume to 50 µl/well with Assay Buffer to generate 0, 2, 4, 6, 8, 10 nmol/well of Triglyceride Standard. For the fluorometric assay, dilute the Triglyceride Standard 10 fold further with the Triglyceride Assay Buffer, then follow the procedure as the colorimetric assay. 3 Lipase: Add 10 µl of lipase to each well with sample and standard. Mix and incubate 10 min at room temperature to convert triglyceride to glycerol and fatty acid. 4. Triglyceride Reaction Mix: Mix enough reagent for the number of samples and standards to be performed. For each well, prepare a total 50 µl Reaction Mix: 46 µl Adipogenesis Assay Buffer 2 µl Probe*** 2 µl Enzyme Mix Add 50 µl of the Reaction Mix to each well containing the Triglyceride Standard, samples and controls. Mix well. Incubate at 37°C for 30 minutes, protect from light. ***
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