Description
Adipolysis Assay Kit (BA0076) (BA0076)
The Adipolysis Assay Kit (SKU: BA0076) provides a rapid, robust and accurate procedure for quantifying adipolysis in cell culture by directly measuring the glycerol released. Obesity develops from the storage of excessive energy as adipose tissue and is a high risk factor for type 2 diabetes, cardiovascular disease and cancer. Adipolysis (lipolysis) is a highly regulated process in fat metabolism in which triglycerides are broken down into glycerol and free fatty acids. This homogeneous assay uses a single working reagent that combines glycerol kinase, glycerol phosphate oxidase and colour reactions in one step. The colour intensity at 570 nm is directly proportional to the glycerol concentration in the sample, and interference from testing drugs is greatly reduced at this wavelength.
| Product Name: | Adipolysis Assay Kit (BA0076) |
| SKU: | BA0076 |
| Detection Method: | Colorimetric or Fluorometric |
| Detection Range: | Colorimetric 0.92 to 100 ug/mL (10 to 1000 uM); fluorometric 0.2 to 5 ug/mL glycerol |
| Sample Type: | Cell culture media (glycerol released during adipolysis) |
| Species Reactivity: | All |
| Assay Time: | 20 minutes |
| Kit Size: | 200 Assays |
| Equipment Required: | Microplate reader |
| Storage: | -20C |
| Shelf Life: | 12 months after receipt |
| Shipping: | Gel Pack |
Quantitative colorimetric or fluorometric assay for adipolysis that directly measures released glycerol. A single working reagent combines glycerol kinase, glycerol phosphate oxidase and colour reactions, read at 570 nm or fluorometrically at excitation/emission 530/585 nm.
- Sensitive and accurate, using as little as 10 uL sample
- Colorimetric linear detection range 0.92 to 100 ug/mL (10 to 1000 uM) glycerol
- Fluorometric linear detection range 0.2 to 5 ug/mL glycerol
- Rapid, convenient single-reagent, 20 minute room-temperature procedure compatible with phenol red media
- Direct assays of adipolysis (glycerol in cell culture media)
- Drug discovery and pharmacology: effects of testing drugs on adipolysis
Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
| Step | Procedure |
| 1 | Cell culture. Grow cells (e.g. preadipocytes, adipocytes) in a culture plate; cells and testing drugs are provided by the user. If desired, treat cells with drugs such as insulin or isoproterenol and incubate. |
| 2 | Standards and samples. Prepare a 100 ug/mL standard by mixing 10 uL 100 mM glycerol standard with 910 uL of the culture medium used, then prepare the dilution series shown in the table. Transfer 10 uL standards into wells of a clear 96-well plate (5 uL for a 384-well plate). Collect cell culture supernatants and transfer 10 uL samples (5 uL for 384-well) into separate wells. |
| 3 | Enzyme reaction. For each well mix 100 uL Assay Buffer, 2 uL Enzyme Mix, 1 uL ATP and 1 uL Dye Reagent. Transfer 100 uL working reagent into each well (50 uL for a 384-well plate) and tap to mix. |
| 4 | Incubate 20 minutes at room temperature and read OD at 570 nm (550-585 nm). If the sample OD exceeds the 100 ug/mL standard, dilute in water and repeat. |
| 5 | Fluorimetric procedure. Dilute standards by mixing 10 uL standard with 190 uL distilled water (final 5.0, 3.0, 1.5 and 0 ug/mL) and dilute cell culture samples similarly (dilution factor n = 20). Transfer 5 uL diluted standards and samples into a black plate, add 50 uL working reagent, incubate 20 minutes at room temperature and read fluorescence at excitation 530 nm and emission 585 nm. |
Subtract the blank OD (standard #4) from the standard OD values and plot against standard concentrations to determine the slope. [Glycerol] = (ODsample - ODmedium) / slope ug/mL. For the fluorimetric assay, [Glycerol] = (Fsample - Fmedium) / slope x 20 ug/mL. Conversion: 1 ug/mL glycerol equals 10.9 uM.
| Component | Quantity | Storage |
| Assay Buffer | 24 mL | -20C |
| Dye Reagent | 220 uL | -20C |
| Enzyme Mix | 500 uL | -20C |
| ATP | 250 uL | -20C |
| Standard (100 mM Glycerol) | 100 uL | -20C |
