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1. Make 4 to 6 pound adult rabbits anemic by subcutaneous injections of 1.2% Acetylphenylhydrazine according to the following schedule:
Day 1: 2 ml
Day 2: 1.6 ml
Day 3: 1.2 ml
Day 4: 1.6 ml
2. Bleed rabbits by cardiac puncture on day 7 or 8 (see Hint #1).
3. Collect 50 to 60 ml of blood from each rabbit into a heparinized syringe and immediately transfer the blood to a 250 ml centrifuge bottle containing an equal volume of saline on ice.
4. Perform all the following steps as rapidly as possible at 4°C. Centrifuge the blood in a GSA rotor at 3500 rpm (2000 X g) at 4°C for 5 min.
5. Aspirate the supernatant and resuspend the cells in 150 ml cold saline. Repeat this washing step three times with a final centrifugation in a GSA rotor at 7000 rpm (8000 X g).
6. Remove as much of the supernatant as possible and estimate the total volume of cells.
7. Lyse the packed cells by the addition of an equal volume of ice-cold ddH2O.
8. Resuspend the cells by pipetting them up and down (see Hint #2).
9. Incubate the cells on ice 1 min and transfer to 30 ml tubes.
10. Centrifuge in an HB4 rotor at 15,000 rpm (37,000 X g) for 20 min at 4°C.
11. Carefully remove the clear supernatant avoiding the large pellet and the fatty surface layer.
12. Rapidly separate the supernatant into 1 ml aliquots and place in liquid nitrogen (see Hint #3 and #4).
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| Acetylphenylhdrazine Solution |
| 1.2% (w/v) Acetylphenylhdrazine
Store at -20°C
Dissolve in ddH2O and neutralize to pH 7.0 with 1 M HEPES, pH 7.0
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| 1 M HEPES, pH 7.0 |
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| Saline |
| 140 mM NaCl
5 mM KCl
1.5 mM MgCl2
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Magnesium Chloride
Potassium Chloride
HEPES
Sodium Chloride
Acetylphenylhyrdazine
Heparin
Liquid nitrogen
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1. Treatment with Acetylphenylhydrazine should greatly increase the percentage of circulating reticulocytes.
2. The solution will become viscous and turn a dark color.
3. The lysate is stable for years if stored beneath the surface of the liquid nitrogen
4. The next step in the procedure is to inactivate the endogenous mRNAs in the lysate so that added message can be efficiently translated (see Protocol for Preparation of Messenger-Dependent Rabbit Reticulocyte Lysate).
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