CELL CENTRIFUGE URGENT!!

[Sarah T.]

If I put in cells that have been broken up into a centrifuge (red meat cells), dose anybody know the levels of seperation ie how much they will seperate? How long would this take in a centrifuge? Can this be done at room temp?

Thanks
Sarah

[mith]

separate what from what?

[canalon]

Depends of the kind of centrifuge, speed, presence of separation medium (fycoll, CsCl,...)

[MrMistery]

also depends on the type of centrifugation technique

[blcr11]

Maybe this will help a little:

http://www.biochemj.org/bj/cp/2006/c010/2006c010.pdf

I don’t do this kind of stuff for a living, so I’m no expert and shouldn’t be taken for one. In principle, I think you can separate various organelles from membranes, nuclei and microsomes, etc. on a single sucrose gradient, but I think what is more typically done is to do graded centrifugations through some kind of stepped sucrose gradient (or ficoll or something like that; and doing everything at 4 C is probably de rigueur). After cracking the cells, the first spin will be relatively low speed to sediment the most dense material (and I don’t know without checking if that would be nuclei or some other structure)—the supernatant would then be diluted a bit and spun a little faster to pellet the next-most-dense fraction, and so on to the least dense. There is a kit (at least one—there may be others) for routine cell fractionation. The kits are commonly used to prepare microsomes to test potential drug candidates for the ability to induce Cyto-P450 in liver cells. (Usually, you’re hoping that the candidate won’t induce since high levels of P450 activity typically means that the compound will have a very high clearance rate and/or high level of conversion to potentially inactive metabolites and so probably won’t be a useful drug, but that doesn’t change the cell-separation principle). You can look at some information on the kit at: http://www.biovision.com/products/k270.html