Description
Fatty Acid Uptake Assay Kit (BA0184) (BA0184)
The Fatty Acid Uptake Assay Kit (SKU: BA0184) provides a fluorescent, cell-based method for measuring long-chain fatty acid uptake and for screening modulators of fatty acid transport. Long-chain fatty acids are important fuel sources and cellular building blocks, and elevated intracellular levels are common in diabetes, obesity-related diseases, cardiovascular disease and certain cancers, making fatty acid uptake a significant therapeutic target. In this assay a fluorescent fatty acid analogue is taken up by fatty acid transporter proteins and accumulates within the cell, while a quench reagent blocks the extracellular fluorescent signal in the medium. As the adherent cells import the analogue, a bottom-read fluorimeter measures the increase in fluorescence at 488/523 nm. This safe, non-radioactive, homogeneous add-and-read assay requires no wash, lysis or staining steps and can be automated for high-throughput fatty acid transport and modulator screens. It is intended for research use only.
| Product Name: | Fatty Acid Uptake Assay Kit (BA0184) |
| SKU: | BA0184 |
| Detection Method: | Fluorimetric cell-based assay (lambda ex/em = 488/523 nm) |
| Sample Type: | Whole cells (e.g. 3T3-L1 adipocytes) |
| Species Reactivity: | All |
| Assay Time: | 60 min read at 37C |
| Kit Size: | 1,000 Assays |
| Equipment Required: | Microplate reader |
| Storage: | -20C |
| Shelf Life: | 12 months after receipt |
| Shipping: | Room Temperature |
A non-radioactive, fluorescent cell-based assay for measuring long-chain fatty acid uptake in whole cells. A fluorescent fatty acid analogue is imported by fatty acid transporter proteins and accumulates intracellularly, while a quench reagent blocks the extracellular signal; a bottom-read fluorimeter measures the increase in fluorescence at 488/523 nm. The homogeneous add-and-read format requires no wash, lysis or staining steps.
- Safe, non-radioactive assay
- Fast and sensitive homogeneous add-and-read assay with no wash, lysis or staining steps
- Simple and convenient; can be automated for high-throughput fatty acid transport and modulator screens
- Determination of long-chain fatty acid uptake in whole cells
- Evaluation of the effects of ligands or drugs on fatty acid transport
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 | Plate 200 uL of 5-8 x 10^4 cells per well in a clear-bottom black 96-well culture plate and incubate for 4 hours or overnight at 37C. |
| 2 | Remove the previous media and starve the cells in 90 uL of serum-free media for 1 hour to increase their metabolic demand. |
| 3 | Add 10 uL of the experimental treatment to the starvation media and incubate at the desired temperature and time (e.g. 37C for 30 minutes), including a solvent control and a blank well with media only. |
| 4 | Prepare Working Reagent 10 minutes before use and warm to 37C by mixing, per reaction well: 1 uL Substrate, 8 uL Quench Reagent and 100 uL Assay Buffer. |
| 5 | Leaving the serum-free media in the well, add an additional 100 uL of Working Reagent to the cells. |
| 6 | Read the plate at 488/523 nm for 60 minutes at 37C using bottom-read settings, and use the data from 60 minutes (F60). |
Compute the mean fluorescence for each treatment by averaging the replicates. Fatty Acid Uptake Activity (%) = [(F_TREATED - F_BLANK) / (F_CONTROL - F_BLANK)] x 100, where F_TREATED is the mean fluorescence of dosed cells, F_CONTROL is the mean fluorescence of undosed cells and F_BLANK is the mean fluorescence of blank (medium-only) wells with no cells.
| Component | Quantity | Storage |
| Assay Buffer | 12 mL | -20C |
| Substrate | 120 uL | -20C |
| Quench Reagent | 1 mL | -20C |
