I'm new to the FRAP assay. When I mixed FeCl3 with TPTZ before adding buffer, I got a dark blue solution instead of a straw-brown solution.

I used the following method:

2 ml of acetate buffer (pH 3.6) + 2 ml TPTZ+ 20 ml of 10 mM ferric chloride solution

(2mL of 40 mM HCl dissolved in 6.25mg TPTZ to prepare TPTZ solution)

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