May 10, 2019
U Mass - Non-esterified fatty acids
- Jason Kim1
- 1University of Massachusetts
- Mouse Metabolic Phenotyping CentersTech. support email: info@mmpc.org
External link: http://mmpc.org/shared/document.aspx?id=168&docType=Protocol
Protocol Citation: Jason Kim 2019. U Mass - Non-esterified fatty acids. protocols.io https://dx.doi.org/10.17504/protocols.io.x3hfqj6
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 12, 2019
Last Modified: May 10, 2019
Protocol Integer ID: 20297
Keywords: Non-esterified fatty acids, obesity, insulin resistance, type 2 diabetes
Abstract
Summary:
This experiment measures serum and plasma concentrations of non-esterified fatty acids using a 96-well kit. The experiment involves a coupled reaction to measure non-esterified fatty acids (NEFA) which ultimately forms a purple product that absorbs light at 550nm. This allows the concentration of NEFA to be determined from the optical density measured at 540~550nm. Serum fatty acids levels reflect systemic lipid metabolism, lipid digestion/absorption, and lipid clearance. Serum fatty acids levels are altered in obesity, insulin resistance, and type 2 diabetes.
Materials
MATERIALS
96-well assay plate blankZen-BioCatalog #SFA-1
Dilution BufferZen-BioCatalog #SFA-1
FFA StandardZen-BioCatalog #SFA-1
FFA Diluent AZen-BioCatalog #SFA-1
FFA Diluent BZen-BioCatalog #SFA-1
FFA Reagent AZen-BioCatalog #SFA-1
FFA Reagent BZen-BioCatalog #SFA-1
Multichannel Pipette TrayZen-BioCatalog #SFA-1
Additional Items
• Multi-channel Pipet , single channel pipet and pipet tips
• Plate reader with a filter of 540 nm
• Incubator at 37ºC
• Tubes for dilution of standards
Reagent Preparation:
Reagent 1:
Preparation of standard curve using the Standard Solution:
1. Briefly spin down the contents of the FFA standard tube before reconstitution.
2. Standard FFA concentrations are 0, 1.4, 4.1, 12.3, 37, 111, and 333 μM.
3. The kit standard solution is the 1.0 mM standard concentration.
4. Pipette 120 μl of Dilution Buffer into 6 tubes.
5. Pipette 60 μl of the FFA Standard Stock solution into a tube labeled 333 μM.
6. Prepare a dilution series as depicted below.
7. Mix each new dilution thoroughly before proceeding to the next solution.
8. The Dilution Buffer alone serves as the zero standard solution.
Reagent 2:
Preparation of FFA Reagent A:
1. Add 10.5 ml FFA Diluent A per bottle, and gently invert. Do not vortex.
2. Store any remaining solution at 2~8°C. The reagent solution is stable for 10 days after reconstitution when refrigerated at 2~8°C.
Reagent 3:
Preparation of FFA Reagent A:
1. Add 5.5 ml FFA Diluent B per bottle, and gently invert. Do not vortex.
2. Store any remaining solution at 2~8°C. The reagent solution is stable for 10 days after reconstitution when refrigerated at 2~8°C.
Before start
Notes:
√ Freshly prepared blood or plasma samples are recommended. If storing samples, keep blood and plasma samples at –20ºC or at -70ºC for long-term storage. Avoid freeze/thaw cycles.
√ Avoid using samples with gross hemolysis or lipemia.
√ Allow all reagents to come to room temperature before measurement.
Add 5 μl (or 1~10 μl) of serum or plasma sample to a well of Plate A.
Add dilution buffer to each well to reach a total sample volume of 50 μl.
Addition of 5 μl results in a 10x dilution of sample (5 μl of serum/plasma sample in 50 μl total sample volume).
Add 50 μl of each standard to empty wells. Use Plate B if necessary.
Add 10.5 ml of the reconstituted FFA Reagent A to one of the disposable trays provided with the kit.
Add 100 μl of FFA Reagent A to each well.
Gently shake the plate to ensure thorough mixing.
Place in a 37ºC incubator for 10 minutes.
Add 5.5 ml of the reconstituted FFA Reagent B to the other disposable tray provided with the kit.
Add 50 μl of FFA Reagent B to each well.
Gently shake the plate to ensure thorough mixing.
Place in a 37ºC incubator for 10 minutes.
Allow the plate to equilibrate to room temperature for 5 minutes. During this time, ensure that there are no bubbles in the solution mixture.
Use a large gauge needle or clean pipet tip to pop any bubbles as this will result in inaccurate absorbance readings.
Measure the optical density of each well at 540 nm.