Biomedical Engineering Reference
In-Depth Information
PROTOCOL 7.4
Enucleation of Micronucleate Cell Populations
Equipment and reagents
•
Percoll (GE; supplied as a sterile suspension in aqueous solution).
•
Cytochalasin B (Sigma) stock solution of 2
μ
g/ml in DMSO.
•
Oakridge, 30 ml polycarbonate tubes (Oakridge Tubes Beckman).
•
High-speed refrigerated centrifuge (Beckman XL-90 ultracentrifuge).
•
Polycarbonate 50ml sterile disposable centrifuge tubes (Corning tubes).
FBS (Wisent)
•
Method
1 Prepare Percoll by adding 92ml of Percoll, 3 ml of 5
M
NaCl and 5 ml of 1
M
Hepes pH
7.0. Add 15ml of equilibrated Percoll to 2
×
30ml Oakridge tubes.
2 Prepare DMEM, supplemented with 10% FBS, adding cytochalasin B to a final
concentration of 20
μ
g/ml.
3 Pellet donor cells by centrifugating at 1000
g
and resuspend in 30ml DMEM, 10% FBS
+
cytochalasin B to a density of
<
3
×
10
7
cells/ml. Clumps of cells should be broken by
trituration.
4 Divide the cell suspension into several tubes with Percoll, filling each to capacity
(approximately 15ml), and mix well by inverting the tubes.
5 Centrifuge the tubes at 31 000
g
in an ultracentrifuge using a 45Ti rotor for 80 min at
20
◦
C.
n
6 After centrifugation, two or more bands should be visible. A sample from about 2 cm
below the top of the tube
o
down to the region just above the Percoll pellet is collected
into 50ml polypropylene tubes.
7 Centrifuge the tubes again at 2000
g
for 10min and remove the supernatant
8 Resuspend and pool the pellets in 50ml of serum-free DMEM and reserve an aliquot for
quantification.
9 Centrifuge pooled pellets at 2000
g
for 10 min and rinse with serum-free DMEM at least
three times to remove Percoll.
Notes
n
It is recommended to fix a low temperature (
∼
20
◦
C) during ultracentifugation to avoid
overwarming that can cause massive cell death.
o
The enucleation process results in extrusion of the nucleus or micronuclei, surrounded by a
small amount of cytoplasm and plasma membrane [36, 37].
