Tissue iron measurement

Procedure

For Liver

1. Homogenize the whole liver (make sure liver samples are in 2ml tubes before proceeding!)
   1. Get two beaks of DI water, homogenization tips, bench pad, Kimwipes, homogenizer, ear plugs
   2. Homogenize til liver turns pink.
   3. Dip tip in one beaker of water for 10 seconds and then the other beaker of water for 10 seconds. Wipe with a kimwipe
2. Pipet 75μL of homogenate in a tared 1.5mL screwcap tube. Record weight
3. Add 1125 μL protein precipitation solution in 1.5 mL and vortex. (See Iron Standard section below)
4. Boil for 30min-1h.
5. Cool in room temperature water for 2 minutes.
6. Open caps to release air bubbles
7. Centrifuge at full speed in microcentrifuge for 10 minutes.

For Spleen

1. Weigh out spleen in a tared 2ml tube. Record weight.
2. Add 1125 μL protein precipitation solution in ea 2ml spleen tube
3. Homogenize the spleen
   1. Get two beaks of DI water, homogenization tips, bench pad, Kimwipes, homogenizer, ear plugs
   2. Homogenize til liver turns pink
   3. Dip tip in one beaker of water for 10 seconds and then the other beaker of water for 10 seconds. Wipe with a kimwipe
4. Boil for 30min-1h. (make sure you put plastic caps on the tubes so the lids do not pop open. They still might so don’t leave it unattended)
5. Cool in room temperature water for 2 minutes.
6. Open caps to release air bubbles
7. Centrifuge at full speed in microcentrifuge for 10 minutes

DCL Iron Kit (also called Iron-SL Assay)

How the kit works:
 

1. Transfer 10 μL of supernatant into 2 wells (as duplicates) of Corning (3604) spectrophotometer plate.
2. Transfer 10 μL of iron standard series and blank into 2 wells of Corning (3604) spectrophotometer plate.
3. Add 170 μL of R1 (acid dissociating reagent)
4. Add 40 μL R2 (color change reagent)
5. Rock for 5 minutes
6. Measure at 595 nm

Iron Standard

Prepare iron standard series by performing 2 fold dilutions of 10 mM Ferric Ammonium Citrate (FW=265 in 10 mL ) DD-H2O water as follows:
- Standards 1-8: 5 – 2.5 – 1.25 – .625–.3125–.15625–.078–.039 mM
- Blank = protein precipitation solution.
You will need 75 μL of each standard to add to 1125 μL of protein precipitation solution in screw cap tubes.

Calculation of the iron concentration

Standard curve (10mM to 0.078mM) => equation y=ax+b where the absorbance value =y
For each absorbance of each sample: x=(y-b)/a and this reflects the iron concentration in 75μL of liver.
x= concentration in mM = mmoles/L=mmoles/kg=umoles/g corresponding to the 75μL of liver

To calculate liver iron:
1μL=1mg. However, 75μL of liver = 30-80 mg

To obtain the iron concentration in umoles/g (1g of liver):
[c] umoles/g= x * (75/recorded weight of the 75μL)
[c] ug/g = [c] umoles/g * 55.5

To calculate spleen iron:
Take the concentration of iron in XμM /(g spleen weight)*(55.50μg/μmol) = μg/g iron in the spleen

Protein Precipitation Solution

.53N HCl and 5.3% trichloroacetic acid in HPLC water. Prepare in fume hood, HCl is extremely volatile.

For: 500 mL: