clogged patch-pipettes

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Simon8
Simon8's picture
clogged patch-pipettes

Hi there!
I used to patch in acute slices and in slice cultures (whole cell), without any unresovled problems. As I now (after a break) moved "back" to dissociated cortical neurons, I was surprised how difficult the switch was. In the meantime I managed to get a few recordings, but still have technical problems.

The most annoying is, that every second (or even more often) pipette gets clogged very fast. I do not need more than a few minutes to approach the cell, but often I can already see that there is something in the tip (a bright dot, as if the pipette would have no opening at all). Then, when trying to get a gigaseal through sucking, no pressure is arriving at the membrane.

Ok here some details what I did to prevent some sources of clogging:
All solutions are sterile-filtered (0,2 µm), the intra even twice, because I use another filter on the filling syringe. To avoid scraping (and thus creating flushable particles) of my Microfil, I firepolished my glasware before pulling the pipettes. I am NOT using positive pressure to approach the cells, this did not prevent clogging anyway.

So my feelings at the moment are, that something is wrong with my intra. Do you think wrong osmolarity could cause this problem? So that something precipitates at the pipette tip? Because I am not used to adjust the osmolarity form my former experience, I did not do it now.

Simon8
Simon8's picture
The problem persisted with a

The problem persisted with a solution of a collegue, who uses to adjust osmolarity.
However, in the meantime, my pipettes do not clog so fast anymore. But I cannot give a reason other than increased speed and security of approaching the cell (so less time or the pipette to get clogged). Although the problem is not completely solved, it is now at least possible fo rme to patch some cells.

I will keep you informed about further insights, if any occur.

Chemmjr
Chemmjr's picture
 I had the same problem.  It

 I had the same problem.  It turned out to be the storage of my glass capillaries.  If your glass comes standing up like mine then I suggest 

1) wear gloves when handling 

2) apply a puff of compressed air through the glass before you fire polish/ pull  (note to much air will cause the glass to chill and negatively affect your pull, so be careful) 

3) clean the outside of the microfil with a kim wipe  

4) before you fill a pipette, point the microfill straight up and allow a drop to fall down the side.  

 

Simon8
Simon8's picture
Thank you very much! Now, my

Thank you very much! Now, my pipettes nearly never get clogged again.

Handling glassware with gloves is something I did before, at least during polishing the ends of the capillary (after that, I take care to not touch the middle of the glas, where the heating filament of the puller will soften the glass). The pressure thing sounds interesting, but i am afrain is not easily possible with our horizontal pragrammable pullers. Your point 4) also sounds reasonable, but I never did that on purpose.

So what is changed now?
I guess, I spend more care on cleaning my microfil before and after my experiment with aqua dest, just by flushing and rinsing. Maybe also the new solution is "better" (I removed the glucose from the extra).

So thank you again. The problem looks as it is solved now!