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== NEFA determination from serum (use Wako Diagnostics NEFA-HR(2) Microtiter Kit) ==
*Note: This page has been updated from a previous version, according The manufacturers protocol says to an update on the manufacturers website. Our previous method utilized 2.5 uL serum/standards in 100 use 225 uL of reaction buffer A and 50 75 uL of reaction buffer Bwith 4 uL of sample, read at 550560:660 670 nm. This uses more reagent than is necessary for the volume of serum/concentrations of NEFA that we would typically analyze.
#To wells of a clear 96 well plate, add 4uL of water (as blank), appropriate standard (the kit comes with stock of 1 mEq/L. Dilute the stock in ddH20 to give 0.25 and 0.5 mEq/L standards. For the high standard, add 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 4 uL of serum samples. ***Note: You may need to add more/less serum, depending on the NEFA concentration of your samples. The assay is linear between 0.01-4.00 mEq/L***
#To each well, add 225 100 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 560:670 660 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 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 75 50 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 560:670 660 nm. These are the final readings.
#Calculations
##For both initial and final readings, subtract the absorbence values obtained at the 670 nm wavelength from those obtained at the 560 nm wavelength.
