Description
Adipogenesis Assay Kit (BA0236) (BA0236)
Adipogenesis is a tightly regulated cellular differentiation process, in which mesenchymal stem cells commit to preadipocytes and preadipocytes differentiate into adipocytes. Adipocytes, holding the largest energy reserve as triglycerol in the body of animals, play a key role in energy homeostasis. An increasingly sedentary lifestyle coupled with an energy-rich diet has contributed to a high frequency of obesity and other health problems such as type 2 diabetes. The Adipogenesis Assay Kit (SKU: BA0236) determines adipogenesis, in which triglycerides are extracted, hydrolysed to glycerol and measured using a Dye Reagent. The colour intensity at 570 nm or fluorescence intensity at 530/585 nm is directly proportional to glycerol concentration in the sample. The single working-reagent, 30-minute room-temperature procedure combines sample extraction, hydrolysis and colour reaction and is amenable to high-throughput screening of adipogenesis modulators.
| Product Name: | Adipogenesis Assay Kit (BA0236) |
| SKU: | BA0236 |
| Detection Method: | Colorimetric (OD570 nm, 550-585 nm) / Fluorimetric (λex/em = 530/585 nm) |
| Detection Range: | Colorimetric: 0.16 to 5 nmoles; Fluorimetric: 0.075 to 0.5 nmoles per 96-well |
| Sample Type: | Cells (e.g. preadipocytes, adipocytes) and tissue |
| Species Reactivity: | All |
| Assay Time: | 30 minutes |
| Kit Size: | 200 Assays |
| Equipment Required: | Microplate reader |
| Storage: | The kit is shipped on ice. Store all components at -20°C. |
| Shelf Life: | 6 months after receipt. |
| Shipping: | Gel Pack |
A quantitative colorimetric/fluorimetric assay for adipogenesis. Triglycerides are extracted, hydrolysed to glycerol and measured using a Dye Reagent; the colour intensity at 570 nm or fluorescence intensity at 530/585 nm is directly proportional to glycerol concentration in the sample.
- Sensitive and accurate. Use as little as 40 µL samples. Linear detection range in 96-well plate: 0.16 to 5 nmoles for colorimetric assays and 0.075 to 0.5 nmoles for fluorimetric assays.
- Fast and convenient. The addition of a single working reagent and 30-min incubation combines sample extraction, hydrolysis and colour reaction. The whole procedure is performed at room temperature with no incubator or heating needed.
- Robust and amenable to HTS. Homogeneous “mix-incubate-measure” type assay. No wash and reagent transfer steps are involved. Can be readily automated on HTS liquid handling systems for processing thousands of samples per day.
- For sensitive quantitative determination of adipogenesis and high-throughput screening of adipogenesis modulators.
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 | Equilibrate all components to room temperature; keep thawed Lipase and Enzyme Mix in a refrigerator or on ice. Avoid SDS and SH-group containing reagents (e.g. mercaptoethanol, DTT) in sample preparation, and include a Sample Blank to correct for background glycerol. |
| 2 | Colorimetric standard curve: prepare 1 mM glycerol standard by mixing 10 µL 100 mM glycerol standard and 990 µL dH2O; dilute to 0.125 mM by mixing 100 µL 1 mM and 700 µL dH2O. Dilute standards in dH2O in wells of a clear 96-well plate (5.0, 3.0, 1.5 and 0 nmol/well per the table). |
| 3 | Sample preparation: cells (10-50 × 10³ preadipocytes/adipocytes) are mixed with 100 µL Extraction Solution, vortexed 30 s and centrifuged 5 min at 14,000 rpm; transfer 40 µL cell lysate to sample wells and 40 µL to a separate Sample Blank well. Tissue (1-5 mg) is homogenised in 100 µL Extraction Solution, or 1-10 µg tissue lysate is mixed with 100 µL Extraction Solution, vortexed and centrifuged; dilute to 25-250 µg/mL and transfer 40 µL Sample per well. |
| 4 | Prepare Working Reagent (WR) per Standard and Sample well: 60 µL Assay Buffer, 2 µL Enzyme Mix, 5 µL Lipase, 1 µL ATP and 1 µL Dye Reagent. Prepare Blank Working Reagent (BWR) per Sample Blank well: 65 µL Assay Buffer, 2 µL Enzyme Mix (no Lipase), 1 µL ATP and 1 µL Dye Reagent. |
| 5 | Transfer 60 µL WR into standards and sample wells and 60 µL BWR into Sample Blank wells only. Tap plate to mix. Incubate 30 min at room temperature. Read optical density at 570 nm (550-585 nm). |
| 6 | Fluorimetric assay: as above but more sensitive, using a black flat-bottom 96-well plate; glycerol standards 0.5, 0.3, 0.15 and 0 nmoles/well. Dilute samples at least 10-fold. Read fluorescence at λex = 530 nm and λem = 585 nm. |
Subtract ODH2O (water, #4) from the standard OD values and plot OD against standard concentrations to determine the Slope by linear regression. [Glycerol] = [(S_Sample – S_Blank) / Slope] × n (nmoles), where S_Sample and S_Blank are the OD or fluorescence intensity values of the Sample and Sample Blank wells and n is the dilution factor. If the Sample OD is higher than the Standard OD at 5 nmoles glycerol, dilute sample in water and repeat the assay, multiplying by n.
| Component | Quantity | Storage |
| Assay Buffer | 24 mL | -20°C |
| Extraction Solution | 8 mL | -20°C |
| ATP | 250 µL | -20°C |
| Dye Reagent | 220 µL | -20°C |
| Enzyme Mix | 500 µL | -20°C |
| Lipase | 1000 µL | -20°C |
| Standard (100 mM Glycerol) | 100 µL | -20°C |
