Laboratory of Molecular Cytogenetics and Cytometry
Institute of Experimental Botany AS CR

Preparation of Fixed Chicken Red Blood Cell Nuclei for Instrument Alignment 
(Dolezel, unpublished)

1. Mix 1 ml fresh chicken blood with 3 ml CRBC buffer I. Centrifuge at 50g for 5 min.
2. Discard the supernatant, add 3 ml CRBC buffer I and gently mix. Centrifuge at 50g for 5 min.
3. Discard the supernatant, resuspend the pellet in 2 ml CRBC buffer II, and vortex briefly.
4. Immediately add 2 ml CRBC buffer III, and mix briefly.
5. Centrifuge at 250g for 5 min. Discard the supernatant, add 2 ml CRBC buffer III, and mix gently.
6. Centrifuge at 120g for 5 min, and discard the supernatant. Transfer the pellet to a clean tube, add 2 ml CRBC buffer III, and mix gently.
7. Centrifuge at 90g for 5 min, and discard the supernatant.
8. Resuspend the pellet, add 2 ml cold fresh fixative (ethanol : acetic acid, 3:1), and vortex briefly.
9. Leave overnight at 4°C, do not shake!
10. Gently remove the fixative, and resuspend the pelleted nuclei.
11. Add 6 ml of ice cold 70% ethanol, vortex briefly, and syringe through a 30G needle, three times.
12. Filter the nuclear suspension through a 42 ľm nylon filter to remove large clumps.
13. Store in aliquots of 2 ml at -20°C. If the concentration of the nuclei is too high, dilute it using ice cold 70% ethanol.

Isolated nuclei can be stored for up to several years without any sign of deterioration. Note: fixed nuclei are not suitable as a standard for estimation of nuclear DNA content in absolute units (genome size).