Protocols


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