Materials and Reagents

1. 1 ml TB syringe with 27-gauge needle (BD Biosciences, catalog number: 309623 )
   
2. Plastic wrap
   
3. 3.5” x 3.5” weigh boat (Thermo Fisher Scientific, Fisher Scientific, catalog number: 8732113 )
   
4. 1.5-ml microcentrifuge tubes
   
5. 0.45 µm filter unit (Thermo Fisher Scientific, Fisher Scientific, catalog number: 09-740-65B )
   
6. Amicon^TM Ultra-15 centrifugal filter units (Merck Millipore, catalog number: UFC903024 )
   
7. 5 ml tube
   
8. Adult mice (> 6 weeks, any strain) (e.g., Balb/cJ)
   
9. ⁵⁹FeCl₃ (Perkin Elmer, catalog number: NEZ037500UC )
   Note: The minimum specific activity of ⁵⁹Fe we have used to obtain decent signal from our gamma counter was 0.2 Ci/mmol at the concentration of ~3 µCi/µl, which gave whole body counts per minute of ~100,000 per each animal. Our gamma counting efficiency is about 10% for ⁵⁹Fe.
   
10. Nitrilotriacetic acid (NTA) (Sigma-Aldrich, catalog number: N9877-100G )
    
11. HEPES (Thermo Fisher Scientific, Fisher Scientific, catalog number: AC172570250 )
    
12. Tris (Thermo Fisher Scientific, Fisher Scientific, catalog number: BP152-5 )
    
13. Sodium bicarbonate (NaHCO₃) (Fisher Scientific, catalog number: S233-500 )
    
14. Sodium citrate dihydrate (Thermo Fisher Scientific, Fisher Scientific, catalog number: S279-500 )
    
15. Ferric chloride hexahydrate (FeCl₃·6H₂O) (Thermo Fisher Scientific, Fisher Scientific, catalog number: I88-100 )
    
16. Hydrochloric acid (HCl) (Thermo Fisher Scientific, Fisher Scientific, catalog number: A144-212 )
    
17. Ethanol
    
18. Human apo-transferrin (Sigma-Aldrich, catalog number: T2252 )
    
19. PBS (VWR, Corning^®, catalog number: 21-040-CM )
    
20. Sodium chloride (NaCl) (Thermo Fisher Scientific, Fisher Scientific, catalog number: S2713 )
    
21. Fisherbrand^TM absorbent underpads (Thermo Fisher Scientific, Fisher Scientific, catalog number: 14-206-64 )
    
22. Radioactive decontaminating solution (Thermo Fisher Scientific, Fisher Scientific, catalog number: NC9633347 )
    
23. 20 mM Fe-NTA (1:4) (see Recipes)
    
24. Ferric citrate solution (see Recipes)
    
25. ⁵⁹Fe-labeled ferric citrate solution (see Recipes)
    
26. ⁵⁹Fe-labeled transferrin (see Recipes)
    
27. 0.9% NaCl solution (see Recipes)
    

Equipment

1. Gamma counter (PerkinElmer, catalog number: 2480-0010 )
   The program for ⁵⁹Fe detection was set using the following parameters:
   1. Counting window: Dynamic-keV
      
   2. Peak position: 1292 keV
      
   3. Low boundary: 1020 keV
      
   4. High boundary: 1400 keV
      
   5. Threshold: 20%
      
   6. Max. assay deviation: 20%
      
   7. Max. normalization deviation: 50%
      
   8. Warning assay deviation: 15%
      
   9. Significant CPM/keV: 10
      
   10. Counting spectrum type: many peaks
       
2. Gamma counting tubes (5 x 28 mm) and RIA racks (PerkinElmer, catalog number: 1480-151 )
   
   1. Prior to gamma counting, empty gamma counting tubes are measured to ensure no ⁵⁹Fe contamination (i.e., < 25 CPM = background).
      
3. Mouse restrainer (Braintree Scientific, catalog number: MTI STD )
   
4. Basic surgical kit for small animals
   

Procedure