Difference between revisions of "Triglyceride Assay from Tissue Culture Cells"

Revision as of 12:33, 29 April 2014

Materials

• Homogenization Buffer (50 mM Tris pH 8, 5 mM EDTA, 30 mM Mannitol, PI inhibitor, can be made in bulk without the PI, PI added fresh)
• 10M KOH
• Chloroform/Methanol Mixture (2:1)
• Butanol Mixture: 3 mL butanol, 1.66 mL Triton-X114, 0.33 mL Methanol
• Sigma Triglyceride Assay Kit (Cat TR0100)

Protocol

1. These volumes are for a well of a 6 well plate.
2. Wash cells with 1 mL of ice cold PBS after treatment
3. Add 200ul Homogenization Buffer
4. Homogenize by sonication (3 x 15s) or 3 x freeze/thaw cycles
5. Add 5ul KOH
6. Mix by inverting
7. Add 800ul Chloroform/Methanol Mixture
8. Vortex vigorously then sit at room temperature for 5 minutes
9. Centrifuge for 10 minutes @ 13000G
10. Transfer the bottom layer into a new tube
11. Let evaporate overnight at room temperature
12. If absorbance is going to be measured by cuvette, use non-bolded values. If using a plate reader, used bolded values.
13. Add 500ul (50ul) of Butanol Mixture. See Suggested Volumes for your specific tissue.
14. Measure triglyceride levels using SIgma Diagnostic Kit, using 5ul of sample.
    1. Resuspend triglyceride and glycerol reagent with water if necessary
    2. Calculate how many sample you have (samples + blank + standard curve)
    3. Prepare reagent. You need 560ul (80ul) of glycerol reagent and 140ul (20ul) of triglyceride reagent. Make extra and combine in a Falcon tube.
    4. Aliquot 700ul into a cuvette or 100ul into a well of a 96 well plate
    5. For standards, add 0-5ul of glycerol standard
    6. Add 5ul of resuspended lipid to each well to start (also make a 5ul blank of the butanol mixture) and mix.
    7. Let sit for ~30 mins @ room temperature (or 5mins @ 37C if you are in a hurry). If using >10ul of butanol mix the solution may be cloudy. Let it settle and it should become more clear.
    8. Measure absorbance @ 540nm
    9. If any samples are A540<0.1 or above the 5ul standard the A540, then repeat with more or less lipid as required.