[Cell-biology] Re: Cell pellets

[Allison]

Kevin Carbajal wrote:
> It seems silly, but I'm having a horrible time pelleting and
> recovering my mouse embryonic stem cells during a wash-instensive
> protocol that labels them for FACS (TUNEL staining and surface marker
> labeling).  I have changed obvious procedures such as centrifugation
> speeds and solution replacing techniques.... but I keep losing too
> many cells during the many washes I have to perform.  The pellets seem
> loosely attached to my tubes... I keep ending up with either full,
> half, or no pellets in my tubes.
> 
> Any ideas on how to make sure I don't lose so many cells... or at
> least how to lose them equally in every tube?  Thanks!
> 

How are you removing the supernatant after spinning?  Can you see your 
cell pellet clearly?

I use vacuum and a pasteur pipette with a yellow Eppendorf tip on the 
end.  The vacuum creates enough suction to 'pick up' the tip from a 
racked box.  Because the hole in the tip is very fine I find it easy to 
aspirate off the supernatant down close to the cell pellet.  And it is 
easier to change the tip between samples than to change pasteur pipettes.
I also use polystyrene tubes instead of polypropylene - the clear walls 
let me see the pellet better.
HTH
Allison