Hi Matthijs, perhaps smaller pipette tips and faster retraction may help?

I sometimes have the same problem and I got the impression that it has to do with how much membrane I suck into the pipette (hard time getting Gigaseal, hard time breaking in). However, I am not certain about this impression.

Which cells are you patching these days? And what is your intrapipette solution?

Edit (28/3): I normally take about 5-10 minutes to close the cell. I recall that others sometimes take 20-30 minutes to close a cell.

Martijn! Nice to hear from you, hope all is well.

Thanks for your advice, I have been using slightly larger pipette tips lately, I’ll attempt with a smaller diameter. In regard of retraction speed, I’ve actually been going for a slow retraction when the nucleus comes along, hoping this will allow the membrane to re-seal again behind the nucleus. This way I may stand a chance at saving the morphology, which should be possible considering that’s what the Patch-seq people are apparently doing. See for instance: Article Electrophysiological, transcriptomic and morphologic profili...

Take care

My impression is that it does not depend on the size of pipette. Rather, it depends on how you approach cell during patching and how strong do you suck in. When you approach cell, you are blowing with solution from the pipette and can see membrane invagination on the cell that you just approached. Try patching just by releasing pressure and waiting 1-2 min for gigaseal without sucking or by applying minimum suction pressure. Then, try to use zap to avoid sucking to break in cell. As for closing cells, in my practice, if nucleus is not attached to pipette, it takes 1-2 min to close the cell. Morphology looked OK after this. Hope, it helps.

Hi Thijs! I would suggest that withdrawing the pipette much faster (in seconds instead of minutes) will prevent formation of a nucleated patch. I used to get nucleated patches and the slower I withdrew the pipette, the better the nucleated patch...!

Have you tried applying just a little bit of positive pressure (10-20 mbar) when you withdraw the pipette? (although you may need to be fast given your biocytin).

Hi Alexey P. Bolshakov, thanks for your input, will try reduce suction even further next time. Must say it will be hard to accept if it really turns out to be down to my patching technique, I’ve been doing it my way for so long; hope I’m not losing my touch…

Hey Mariana Vargas-Caballero, good to hear from you and thanks for the pro tips! Regarding retraction speed, I too prefer a relatively fast retraction over a couple of seconds, starting slowly and gradually increasing pace if you see your cell is closing nicely on the membrane seal test. This has always worked fine for me; it’s just recently that I’ve been having the issue of sticky nuclei. A bit of positive pressure only helps occasionally sadly… When the lab reopens from the corona lock-down I’ll review everything and hope I find the culprit. If so, I’ll be sure to report back here. Be well.

Hi!

Our lab lately seems to have a somewhat similar problem.

My patches tend to cIose again shortly after break in, because of the nucleus getting sucked towards the patch pipette. when i retract my pipette, i can see the nucleus attached. As it is often is not easy to seal either, using less negative pressure wont help either. We are patching in organotypic slice cultures and use potassiumgluconate or potassiumchloride based internals. With the latter, the problem seems to be even worse...

Any ideas are very welcome!

We are having the feeling that it could be due to osmolarity, as we realized the osmolarity of the culture medium is increasing quite quickly (30-50 mOsm in 48 hours), but more medium changes dont seem to help...

Thanks!