Adipolysis Assay Kit, BioAssay™
|Kits and Assays||Storage: 4°C/-20°CShipping: Dry Ice|
Obesity is a chronic condition that develops from storage of excessive energy in the form of adipose tissue. The resulting adiposity presents a high risk factor for diseases such as type 2 diabetes, cardiovascular diseases, and cancer. ADIPOLYSIS or lipolysis is a highly regulated process in fat metabolism, in which triglycerides are broken down into glycerol and free fatty acids. Rapid, robust and accurate procedures for adipolysis quantification in cell culture are very useful in research and drug discovery. Adipolysis assay kit directly measures glycerol released during adipolysis. This homogeneous assay uses a single Working Reagent that combines glycerol kinase, glycerol phosphate oxidase and color reactions in one step. The color intensity of the reaction product at 570nm is directly proportional to glycerol concentration in the sample.
Sensitive and accurate. Use as little as 10ul samples. Linear detection range in 96-well plate: 0.92 to 100 ug/mL (10 to 1000uM) glycerol for Colorimetric Assay:s and 0.2 to 5 ug/mL for Fluorometric Assay:s.
Rapid and convenient. The procedure involves addition of a single working reagent and incubation for 20 min at room temperature.
Robust and amenable to HTS assays. Potential interference by testing drugs is greatly reduced at 570nm. Compatible with culture media containing phenol red. Assays can be performed in 96 or 384-well plates.
Direct Assays: adipolysis (glycerol in cell culture media).
Drug Discovery/Pharmacology: effects of testing drugs on adipolysis.
Kit Contents: (bulk reagents available)
Assay Buffer: 24ml Enzyme Mix: 500ul ATP: 250ul
Dye Reagent: 220ul Standard: 100ul 100mM Glycerol
Storage conditions. The kit is shipped on ice. Store Assay Buffer at 4°C and other reagents at -20°C. Shelf life of 6 months after receipt. Precautions: reagents are for research use only. Normal precautions for laboratory reagents should be exercised while using the reagents. Please refer to Material Safety Data Sheet for detailed information.
SH-group containing reagents (e.g. mercaptoethanol, DTT) may interfere with this assay and should be avoided in sample preparation. Prior to the assay, equilibrate all components to room temperature. Keep thawed Enzyme Mix in a refrigerator or on ice during assays.
1. Cell Culture. Note: Cells and testing drugs are to be provided by the customer and are not included in this reagent kit. Grow cells (e.g. preadipocytes, adipocytes) in culture plate (24-well, 96-well or 384-well). If desired, treat cells with testing drugs such as insulin, isoproterenol, and incubate for the desired time period.
2. Standards and Samples. Prepare a 100ug/mL standard by mixing 10ul 100mM glycerol standard with 910ul in the same medium used for cell culture. Dilute standard in the medium as follows. Transfer 10ul standards into wells of a clear 96-well assay plate (5ul for 384-well assay plate).
No 100 ug/mL STD+Medium Vol (ul) Glycerol (ug/mL)
1 400ul+0ul 400 100
2 300ul+200ul 500 60
3 150ul+350ul 500 30
4 0ul+500ul 500 0
Collect cell culture supernatants from culture wells. Such samples should be assayed immediately or stored at -20°C. Transfer 10ul samples (5ul for 384-well assay plate) into separate wells of the assay plate.
3. Enzyme Reaction. For each assay well, mix 100ul Assay Buffer, 2ul Enzyme Mix, 1ul ATP and 1ul Dye Reagent in a clean tube. Transfer 100ul Working Reagent into each assay well. Tap plate to mix. For assays in a 384-well plate, use 50ul Working Reagent per well.
4. Incubate 20 min at room temperature. Read optical density at 570nm (550-585nm).
Note: if the Sample OD is higher than the Standard OD at 100 ug/mL, dilute sample in water and repeat the assay. Multiply result by the dilution factor.
Subtract blank OD (#4) from the standard OD values and plot the OD against standard concentrations. Determine the slope using linear regression fitting. The glycerol concentration of Sample is calculated
ODSAMPLE and ODMEDIUM are optical density values of the sample and
medium (#4). Conversions: 1 ug/mL glycerol equals 10.9uM.
For Fluorometric Assay:s, the linear detection range is 0.2 to 5 ug/mL glycerol. Dilute Standards (#1 to # 4, see Colorimetric Procedure:) as follows: mix 10ul standard with 190ul dH2O. The glycerol concentrations are now 5.0, 3.0, 1.5 and 0 ug/mL, respectively. Cell culture supernatant: dilute by mixing 10ul cell culture sample with 190ul dH2O (dilution factor n=20). Transfer 5ul of the diluted standards and samples into separate wells of a black 96-well or 384-well plate. Add 50ul Working Reagent and tap plate to mix. Incubate 20 min at room temperature and read fluorescence at lex=530nm and lem=585nm. The glycerol concentration of Sample is calculated as
x 20 (ug/mL)
Materials Required, But Not Provided:
Pipeting devices, centrifuge tubes, appropriate 96- or 384-well plates and plate reader.
Important Note: This product as supplied is intended for research use only, not for use in human, therapeutic or diagnostic applications without the expressed written authorization of United States Biological.