1.Mix 1 ml fresh chicken blood with 3 ml of CRBC buffer I. Centrifuge at 50g for 5 min.
2.Discard the supernatant, resuspend the pellet in 2 ml of CRBC buffer I and mix gently. Centrifuge at 50g for 5 min.
3.Discard the supernatant, resuspend the pellet in 2 ml of CRBC buffer II, and vortex briefly. Immediately add 2 ml of CRBC buffer III, and mix briefly.
4.Centrifuge at 250g for 5 min. (Alternatively, centrifuge at 200g for 5 min).
5.Discard the supernatant, add 2 ml of CRBC buffer III, and mix gently.
6.Centrifuge at 120g for 5 min. (Alternatively, centrifuge at 100g for 5 min).
7.Discard the supernatant, add 2 ml of CRBC buffer III and mix gently.
8.Filter the suspension through a 42 um nylon filter into clean tube, to remove large clumps. Centrifuge at 90g for 5 min. (Alternatively, centrifuge at 70g for 5 min).
9.Discard the supernatant, add 2 ml of CRBC buffer III, mix gently and centrifuge at 90g for 5 min. (Alternatively, centrifuge at 70g for 5 min).
10.Discard the supernatant, vortex the pellet gently, and add 2 ml of cold fresh fixative (ethanol:acetic acid, 3:1). Vortex briefly.
11.If the large clumps occurred in the suspension, filter it through a 42 um nylon filter into clean tube.
12.Leave overnight at 4°C, do not shake!
13.Gently remove the fixative, and vortex the pellet gently.
14.Add 6 ml of ice cold 70% ethanol (if the pellet is weak, add 3 ml of ice cold 70% ethanol), vortex briefly, and syringe through a 30G needle for three times.
15.Finally, filter the nuclear suspension through a 42 um nylon filter to remove large clumps.
16.Store at -20°C.
17.If the concentration of the nuclei is too high, dilute it using ice cold 70% ethanol.
