Need help: patch clamp with marin Hydropolyp

Dr. Alex aledain at receptor.pharm.uwa.edu.au
Tue Apr 16 23:11:23 EST 1996


In article <4l00nc$rma at majestix.uni-muenster.de>,
   Thiemo Sieger <sieger at uni-muenster.de> wrote:
>It is now for months, that I've tried to get seals with the 
Cnidocytes of 
>Cladonema, a marin Hydropolyp.
>I've tested many different bath-solutions (mainly reduction of > 
Cl-),  .... snip problems ...

G'day. Sometimes certain preps are really hard to deal with, and I 
don't know enough about this particular prep to offer any advice 
about it specifically. However, in general terms, maybe these 
wuick ideas will help.

The first thing I would try is increasing the Mg or Ca concentration 
(up to 40-50mM). This is high, but salt bridging is important in 
seal formation. If this is succesfull then you can slowly reduce the 
concentration to a more reasonable level. I presume all the 
solutions you have are filtered (a dumb question), but sometimes 
when you add a cell suspension etc to your bath you introduce a lot 
of floating debris that affects seal formation, try flushing the 
bath after you've "blown" away the debris from the cell surface. 
Then of course another resort is mild enzymatic treatment -  a 
contradiction in terms if ever I've heard one. But a couple I have 
used with success are trypsin or papain, both available from Sigma.
Again another technique is to actually fire-polish the pipette tip. 
For many preps and the new beaut pipette pullers they have nowadays 
it's not necessary to fire-polish, but it may just give you that 10% 
extra success rate (I used to have to pipette polish for one prep, 
but not for another?).

Hope these ideas help, feel free to email me for further advice.

cheers, Alex.

----------------------------------------------------------
Dr. A. C. Le Dain
Department of Pharmacology
University of Western Australia
Ph (09) 346 3650
Fx (09) 346 3469
aledain at receptor.pharm.uwa.edu.au
----------------------------------------------------------





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