Changes

***Note: This page has been updated from a previous version, according to an update on the manufacturers website. Our previous method utilized 2.5 uL serum/standards in 100 uL reaction buffer A and 50 uL reaction buffer B, read at 550:660 nm***#To wells of a clear 96 well plate, add 2.5 uL 4uL of water (as blank), appropriate standard (the kit comes with stock of 1 mEq/L. Dilute the stock in ddH20 to give 20.5 25 and 0.5 mEq/L standards. For the high standard, add 5 8 uL of the 1 mEq/L stock. Due to the extra volume, the concentration of this last standard is actually 1.97 mEq/L).#To remaining wells, add 2.5 4 uL of serum samples.***Note: You may need to add more/less serum, depending on the NEFA concentration of your samples***#To each well, add 100 225 uL of reaction buffer A, mix gently and incubate at 37 deg C for ~5 minutes. #Allow plate to return to room temperature before reading at 550560:560 670 nm. These are the initial readings. ##Note: If the plate reader being used does not allow for measuring two wavelengths simultaneously, read the plate at 550 560 nm. The rationale behind measuring two wavelengths simultaneously is to account for possible sample contamination by products of haemolysis, which can interfere with the assay. #To each well, add 50 75 uL of reaction buffer B and incubate at 37 deg C for ~5 minutes. The wells will turn purple. If they do not, check whether the reagents are within the best before date.#Allow plate to return to room temperature before re-reading at 550:560 :670 nm. These are the final readings.

##For both initial and final readings, subtract the absorbence values obtained at the 560 670 nm wavelength from those obtained at the 550 560 nm wavelength.