Biomedical Engineering Reference
In-Depth Information
tube, e.g., for Beckman Coulter NVT 65.2 rotor, and centrifuge at
350 000
g (18 900 rpm)at20
◦
C for 6 h.
After the run, unload the tube either by puncture of the thin-
walled tube with a syringe needle at the bottom, or by displacement
by 80% cesium chloride (w/w). For DNA content, read UV absorp-
tion of the fractions and refractive index for density calculation
(for equations see footnote to Table 5.4).
The content of guanine G and cytosine C of double-stranded
DNA is calculated from density according to Schildkraut et al.:
×
ρ
(
− 1.66)
·
100
=
Mol − %GC
0.098
References
Osterman LA (1984) Methods of protein and nucleic acid research, vol. 1:
Electrophoresis, isoelectric focusing, ultracentrifugation. Springer,
Berlin, p. 284
Schildkraut CL, Marmur J, Doty P (1962) J Mol Biol 4:430
5.3.5.2 Cell Fractionation Using Percoll
Percoll is a polyvinylpyrrolidone-coated silica sol with very low
osmolarity. For cell separations therefore the osmolarity is adjusted
to isotonic conditions by mixing 9 vol. Percoll stock suspension
with 1 vol.of2.5M sucrose, 1.5 M sodium chloride, or tenfold
concentrated cell culture medium (e.g., 10
RPMI).
Prepare the density gradient either stepwise or continuously;
generation of the gradient by centrifugation is also possible. In
comparison with cesium chloride gradients, the Percoll gradient
forms fast during centrifugation.
×
A .5M sucrose or 1.5 M NaCl or tenfold concentrated cell cul-
Solutions/Reagents
ture medium
B1vol. Soln. A and 9 vol. ddH
2
O
C Percoll working dilution: dilution of Percoll with a density
nearbythedensityofthecellstobeisolated;toprepareVml
of working dilution mix V/10 ml of Soln. A with V
0 ml
Percoll
stock
5
and (V − V
0
) ml ddH
2
O
For preparation of a density gradient with a large linear range,
manufacturers give the following conditions: Spin Percoll in 0.15 M
NaCl (final concentration) with at least 10 000
×
g
av
,forPercollin
0.25 M sucrose apply 25 000
×
g
av
. Use near vertical or vertical
rotors for cell separation.
5
=
ρ
·
ρ
A
− 0.9)
/
ρ
0
;V
0
,mlPercoll,V,finalvolumeof
V
0
V
·
(
− 0.1
ρ
ρ
0
,density
working dilution,
, required density of working dilution,
/
ρ
of Percoll stock solution (
≈
1.13 g
ml),
A
, density of Soln. A (1.5 M
/
/
NaCl: 1.058 g
ml;2.5M sucrose: 1.316 g
ml)
