Inert "carrier" for small numbers of tissue culture cells duringcentrifugation?

GysdeJongh via methods%40net.bio.net (by jongh711 from planet.nl)
Mon Oct 6 05:14:19 EST 2008


"Wendy Ingram" <w.ingram from uq.edu.au> wrote in message 
news:mailman.256.1223240385.29717.methods from net.bio.net...
> Does anyone know of an inert substance, home made or commercial, that
> can be added to mammalian cell cultures when spinning? I'm looking for a
> "carrier" that won't harm live cells, but provides "bulk" to the cell
> pellet when it would otherwise be very tiny. The goal is to increase
> recovery when there are very few cells... Hopefully using something that
> looks different to cells under the microscope when pellets are
> eventually resuspended. I guess I'm looking for the live cell equivalent
> of "pellet paint", which is used as a carrier for DNA work. Does anyone
> know if there is there something similar out there for tissue culture use?

Hi Wendy Ingram,
surprising question.I just wonder why do you want to do this ?? I did a lot 
of cell culture in my career and I frequently purified low numbers of 
cells.From FAC's experiments for instance.

Here are a few tricks :
I found that the biggest problem is the plastic tube and just the mechanical 
mixing.Cells tend to stick to plastic irreversibly and are whirled up easily 
because they are much heavier than water.

1) Use conical centrifuge tubes, this will prevent un-intended re-mixing
2) Cool all media to 4 degrees before you start.The sticking of cells is 
temp dependant
3) Rinse  _every thing_  you are going to use with a cold solution of about 
1% bovine serum albumine or, even better, fetal calf serum.Also plastic 
pipets
4) The weight of the cells is much higher than water; they are very fragile 
after all the previuous maniulations.Use the lowest centrifugal force that 
works for you.Or increase the time first and not the rpm.I used 200 g for 10 
minutes.
5) Never aspirate  _all_  of the supernatant.If you want thorough washing 
than increase the number of washing steps.
6) Never use a vortex.It will cause clumbs.Use a disposable (pre-rinsed !!!) 
plastic pipet and position it  _above_  and not just  "_in_ " your 
(supposed) pellet.Then very slowly move the supernatant up- and down.
7) If you need the precise volume do this by weighing the tube with- and 
without your cell suspension.Don't remove all the supernatant just for this 
pupose.
8) A Burker chamber with 0.5 microliter of the cell suspension can be used 
to do a quick check on the quality of the cells under a phase contrast 
microscope at a 25 magnification.

hth
Gys




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