microinjection feasibility questions
pterm at karsh.demon.co.uk
Tue Apr 29 14:46:31 EST 1997
In article <rfischer-2804971842390001 at mquinlan3.utmem.edu>,
rfischer at utmem1.utmem.edu (Bob Fischer) wrote:
> I'm considering a project using epithelial cells from primary cultures
> that would require the microinjection of plasmids, mAbs, and purified GST
> fusion proteins. Anyone know how feasible it would be to microinject
> epithelial cells that must grow in contact with each other? I realise that
> microinjection has been done with NRK cells, retinal pigment cell types,
> etc., but these seem to survive seeding at low cell densities, something
> that primary cells, explant cells, etc. do not deal well with! Also, how
> many cells could be realistically injected in a day, given a relatively
> small cell cytoplasmic area/volume and moderate expertise? Are there
> commercial sources for "pre-pulled" microcapillary tubes?
> Thanks very much in advance.
Cells grown at high density should not necessarily be more difficult to
inject. In fact, you will waste less time moving from one cell to the
next that way! One difficulty, however, may be that you cannot hope to
inject every cell on your coverslip/dish as you may be able to do with
very low density cultures, so you will need a reliable method of
identifying cells that have received injections.
Once you have gained the necessary expertise, you should be able to make
thousands of injections in a day. However, be warned that in a situation
like that, it is not expertise but rather your capacity for boredom that
is the limiting factor!
If you have lots of money to spend, you might consider buying one of the
Eppendorf automatic injections systems. Eppendorf also make pre-pulled
micorcapillaries ("femto-tips"). I have used these extensively with
success, but I know others who find they damage the cells too much. Best
approach might be to see if a rep will give you a free sample pack.
I have published several research papers using intracellular
microinjection into neurons and a couple of review articles on the
technique. See http://www.ed.ac.uk/~mulderry/Pages/pubs.html
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