Publicidad
Methodology Guidelines
94
Manual de operaciones de la Cytospin 4
relatively large fluid volumes, many with relatively few cells.
Such specimens must obviously be concentrated prior to use
of the Cytospin. Such concentration requires the use of a
general laboratory centrifuge. The amount of the specimen
submitted will determine the size centrifuge tubes that
will be necessary. In some cases, the original specimen may
need to be split between many tubes. As an example, if a
centrifuge is available which can hold 50 ml tubes, and the
total amount of specimen is 100 ml or less, then two tubes
can concentrate the entire specimen. Should the specimen
amount to 150 ml, then four tubes would be required. By
adding more tubes it is possible to concentrate specimens
which are quite large in volume. To sediment the cells, the
centrifuge should be spun at 2000 to 3000 rpm for 10 to 20
minutes. Avoid spinning at excessive speeds. This will only
damage cells, and pack them into such tight buttons they
will be difficult to process further. Centrifuges with swinging
buckets will generally require slightly higher speeds than
angle headed centrifuges.
After conventional centrifugation, any cells present in the
sample would appear as a packed button in the bottom of
the tube. The clear supernatant above the cell button should
be carefully aspirated or poured off, leaving a volume of fluid
approximately equal to the volume of the packed cell button.
The fluid that is aspirated or poured off may be discarded,
using any common technique to render it harmless
(sterilisation, fixation etc). This fluid should only be
discarded after it is determined that the cells within have
indeed been retained.
The packed cell button in the bottom of the centrifuge tube
should be thoroughly mixed with the residual fluid that was
not removed. This is done either by use of a vortex mixer, or
by gentle agitation of the tube. The result is a concentrated
cell suspension, suitable for cell number determination, and
subsequent preparation with the Cytospin.
A78310250ES 5.1ª Edición
Publicidad
