TITLE: Antioxidant activity by FRAP assay: in vitro protocol
AUTHORS: Adrieli Sachett, Matheus Gallas-Lopes, Greicy M M Conterato, Ana Herrmann, Angelo Piato

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Considering the role of oxidative stress in the pathology of several diseases and the use of antioxidants as treatment and/or adjuvants in these conditions. Here we propose a protocol to evaluate the antioxidant capacity of compounds by the FRAP method, which is based on the reduction of Fe3+ (ferric) to Fe2+ (ferrous) by antioxidant molecules forming the blue-colored Fe2++ TPTZ complex. This protocol was standardized at LAPCOM (Psychopharmacology and Behavior Laboratory at UFRGS) to assess biochemical parameters </span><span style = "font-style:italic;">in vitro.</span></div><div style = "text-align :; float : ;"><img style = "" src = "https://lh4.googleusercontent.com/52v_JFz8uUrDzcu7EL_d_WJh_lL0y6tqyWGRQ_sfhr-t8Ru3miSCK5ce9Aa1LR2ODmvZ9yIVBZw5VhscsK7PoTg7FUBnm-Hq_WDhCkAKqdWfVsIETzGAVDajfyba0qKCK7-gdNniQos" /></div></div>

[STEPS]
?. [Preparing the reagents]
The first step is to prepare the reagents to be used in this protocol;
?. [Preparing the reagents]
Acetate buffer :     1.1.1 Weigh  of sodium acetate in a beaker of appropriate size;    1.1.2 Dissolve the salt with  of ultrapure water;    1.1.3 Transfer the solution to a  volumetric flask;    1.1.4 Add slowly  of glacial acetic acid to the volumetric flask;    1.1.5 Using ultrapure water, complete the solution's volume to reach ;    1.1.6 Use a pH sensor to evaluate your solution. Expected conditions:;    1.1.7 Store this solution in an amber flask at ;
0.19 g
10 mL
100 mL
1.6 mL
100 mL
8 °C
?. [Preparing the reagents]
2,4,6-Tris(2-pyridyl)-s-triazine (TPTZ)  + Hydrochloric acid (HCl) :     1.2.1  Pepare a solution of HCl  using HCl ;    1.2.2  Fill a  volumetric flask with  of ultrapure water;    1.2.3  Add  of HCl  to the volumetric flask;    1.2.4  Using ultrapure water, complete the solution's volume to reach . (This solution is suited for use for 2 days);    1.2.5  Weigh  of TPTZ in a beaker of appropriate size;    1.2.6  Add  of HCl ;    1.2.8  Transfer the solution to a  volumetric flask;    1.2.8  Using HCl  , complete the solution's volume to reach ;    1.2.9  Store this solution in an amber flask at ;
10 mL
5 mL
40 µl
10 mL
0.031 g
8 mL
10 mL
10 mL
8 °C
?. [Preparing the reagents]
Iron(III) chloride hexahydrate (FeCl3.6H2O) :     1.3.1 Weigh  of ferric chloride Iron(III) chloride hexahydrate;    1.3.2 Transfer the Iron(III) chloride hexahydrate to a beaker of appropriate size;    1.3.3 Add  of ultrapure water to the beaker to dissolve the salt;    1.3.4 Transfer your solution to a  volumetric flask;    1.3.5 Using ultrapure water, complete the solution's volume to reach ;     1.3.6 Prepare this solution on the day of the experiment;
0.0541 g
5 mL
10 mL
10 mL
?. [Preparing the reagents]
Iron(II) sulfate heptahydrate (FeSO4.7H2O) :    1.4.1 Weigh  of Iron(II) sulfate heptahydrate;    1.4.2  Transfer the Iron(II) sulfate heptahydrate to a beaker of appropriate size;    1.4.3 Add  of ultrapure water to the beaker to dissolve the salt;    1.4.4 Transfer your solution to a  volumetric flask;    1.4.5 Using ultrapure water, complete the solution's volume to reach ;     1.4.6 Prepare this solution on the day of the experiment;
0.0556 g
90 mL
100 mL
100 mL
?. [Working solutions]
These solutions should be prepared only on the day of the assay by mixing the previously described reagents. You should mix the solutions in a container of appropriate size wrapped in aluminum foil. Keep the solutions on ice.
?. [Working solutions]
Working solution without TPTZ (perform this step only if the sample has color):     Mix the following reagents in a proportion of 10:1:1Acetate buffer ; Ultrapure water ; Iron(III) chloride hexahydrate (FeCl3.6H2O) ;
15 mL
1.5 mL
1.5 mL
?. [Working solutions]
Working solution with TPTZ:    Mix the following reagents in a proportion of 10:1:1Acetate buffer ; TPTZ solution ; Iron(III) chloride hexahydrate (FeCl3.6H2O) ;
15 mL
1.5 mL
1.5 mL
?. [Incubation of the samples]
To optimize the reaction, an incubation step is needed.
?. [Incubation of the samples]
Prepare  microtubes, to be used to store the samples, with the correct information. The number of microtubes depends on the number of samples. You should provide at least five replicates (n = 5) of each sample with at least one control tube per sample. You should also prepare negative and positive control samples. Perform the test at least two times to ensure the results are as correct as possible.
1.5 mL
?. [Incubation of the samples]
To generate the standard curve, fill the wells of your microplate as described below. You should provide duplicates or triplicates of each point of the curve to make your quantification more precise. Using a micropipette fill the wells in this order: Iron(II) sulfate heptahydrate (FeSO4.7H2O) + Ultrapure water + Working solution with TPTZ.  Air bubbles should be perforated with a needle to avoid bias in the analysis;Microtube concentration of FeSO4.7H2O FeSO4.7H2O (µL)Ultrapure water (µL)Working solution with TPTZ (µL)0-103001000.59.53004002830080046300120064300160082300200010-300
Microtube concentration of FeSO4.7H2O FeSO4.7H2O (µL)Ultrapure water (µL)Working solution with TPTZ (µL)0-103001000.59.53004002830080046300120064300160082300200010-300
?. [Incubation of the samples]
For each sample, fill the plastic microtubes as described below. Using a micropipette fill the tubes in this order: Sample + Ultrapure water + Working reagent without TPTZ or Working reagent with TPTZ. Mix the solution with the pipette tip to homogenize the content; MicrotubeSample (µL)Ultrapure water (µL)Working reagent without TPTZ (µL)Working reagent with TPTZ (µL)Blank-10-300Sample blank10-300-Sample10--300
MicrotubeSample (µL)Ultrapure water (µL)Working reagent without TPTZ (µL)Working reagent with TPTZ (µL)Blank-10-300Sample blank10-300-Sample10--300
?. [Incubation of the samples]
Incubate all your microtubes at  for  using a dry bath;
37 °C
?. [Incubation of the samples]
Take your samples off the dry bath and let them rest at room temperature for ;
?. [Reading your samples]
Prepare to read the absorbance of your samples in a microplate reader;
?. [Reading your samples]
Use a conventional 96-well microplate to run your samples. Before start pipetting, each well of the microplate should be marked for sample identification.
?. [Reading your samples]
Transfer  of the content of each microtube to its corresponding well in the microplate;
250
?. [Reading your samples]
Read the absorbance of the samples at  in a microplate reader;
593
?. [Calculating data and determinig results]
Prepare to analyze the results obtained after reading the absorbance of the samples;
?. [Calculating data and determinig results]
Subtract the absorbance of the blank wells from the absorbance of the different FeSO4.7H2O concentrations:Absorbance of FeSO4.7H2O = Absorbance of FeSO4.7H2O - Blank absorbance
?. [Calculating data and determinig results]
Plot in an excel sheet the results from your standard curve. Ferrous sulfate (FeSO4.7H2O) concentrations in µM are plotted on the X axis, while the respective absorbances (Sample absorbance - Blank absorbance) Should be plotted on the Y axis (as shown in the figure below). Insert a scatter plot, add a trend line, line equation, and R² value. The value of R² must be between 0.9 and 1.
?. [Calculating data and determinig results]
Subtract the absorbance of the sample blank wells from the absorbance of the different samples:Sample absorbance = Sample absorbance - Sample blank absorbance
?. [Calculating data and determinig results]
From the line equation, replace the Y with the sample absorbance (Sample absorbance - Sample blank) and calculate the X value, resulting in Fe2+ concentration in micromoles per liter (µM) in the analyzed sample.
?. [Calculating data and determinig results]
Calculate the amount of Fe2+ in micromoles (µmol) in the final volume of the microtube:Microtube volume: Fe2+ concentration found in the lat step ---------- 1000 mLY                                       --------------------   0.31 mLY = ......  equivalent µmoles of Fe2+
0.31 mL
?. [Calculating data and determinig results]
Calculate the amount of Fe2+ per mL of sample used in the technique:Y found in the last step  ------------ 0.01 mLZ                     ------------------   1 mLZ = ......  equivalent µmoles of Fe2+ / mL of sample
?. [Calculating data and determinig results]
Results should be expressed as equivalent µmoles of Fe2+/mL of the sample.