Tissue iron measurement
Procedure
For Liver
- 1. Homogenize the whole liver (make sure liver samples are in 2ml tubes before proceeding!)
- a. Get two beaks of DI water, homogenization tips, bench pad, Kimwipes, homogenizer, ear plugs
- b. Homogenize til liver turns pink.
- c. 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 75uL 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
- a. Get two beaks of DI water, homogenization tips, bench pad, Kimwipes, homogenizer, ear plugs
- b. Homogenize til liver turns pink
- c. 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 ul of R1 (acid dissociating reagent)
- 4. Add 40 ul 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 ul of each standard to add to 1125 ul 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 75uL of liver.
x= concentration in mM = mmoles/L=mmoles/kg=umoles/g corresponding to the 75uL of liver
To calculate liver iron:
1uL=1mg. However, 75uL 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 75uL)
[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:Substance | Concentration | Volume/Weight | Final Conc. |
---|---|---|---|
HCI | 12 N | 22 ml | 0.53 N |
TCA | 26.5 g | 5.3% M | |
ddH2O | 55 M | Bring volume to 500 ml |