[Cell-biology] Re: separation of cells from supernatants

Bob bbx107 at excite.XXXX.com
Thu Sep 29 22:06:12 EST 2005


On 29 Sep 2005 07:53:43 -0700, murali.subramanian at gmail.com wrote:

>i have tried sucrose gradient centrifugation but may try it again. are
>there any good protocols you have used in the past?

Murali,

Common sense says that the cells are more dense than the oil droplets.
That was the basis of Eric's suggestion. Do you have any reason to
believe otherwise? 

Now you say that sucrose gradient did not work; apparently both stayed
at top. So the obvious conclusion is that you used too much sucrose.
Why not just spin down the cells in "water" (or buffer or growth
medium, or whatever)? If your intent is to throw them out, what is
wrong with pelleting them? 

Do you understand how centrifugation works -- how it depends on the
relative density of the medium and of the molecules (particles) being
centrifuged??

Good protocols from the past are not your solution. You have said that
your application is unusual. Try something, and if it doesn’t work
adjust the procedure, using your understanding of centrifugation. The
only real barrier I would foresee is if the cells are less dense than
water. That seems unlikely. But if it were true, then you could do
reverse centrifugation... put the sample at the bottom, and let it
come up. In fact, if the oil droplets are large enough to float up
fairly rapidly upon centrifugation, that might be an interesting thing
to try.

Are you sure that the cells and oil droplets are not bound to each
other??


bob




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