I did patch CHO cells when I was in S. Korn Lab (UCONN). I didn’t stay long, but his students: P. Andalib, J. Trapani and J. Consiglio were using them routinely with HEK293 too without any particular problem. I suggest you google Trapani’s name along with CHO and look at some of their work regarding Kv channels. Good luck!

Very flat cells and indeed difficult to patch. As with other cultured cells, it is important that the membranes are healthy, so first tip would be to be extra careful when preparing them to patch. This is different from cultured neurons, which normally have plenty of time to recover. Optimize method and timing of cell splitting and recovery, prior to patching. Aim for the nuclear area. Polished pipettes would be ideal, gentle postitive pressure on approach and then just release the pressure, they should seal pretty much on their own. If you are plating them on coverslips, I would fix the coverslip to the chamber (eg with some dow corning grease) to ensure maximum stability. Good luck!

@Fouad Lemtiri-Chlieh

Thanks, I'll take a look at the papers.

@Marisol Sampedro Castaneda

Thanks for all info! Our protocol is:

Day 1 - Split cells onto PDL-coated glass coverslips.

Day 2 afternoon - transfect

Day 3/4 - patch

Does that sound sensible?

I sometimes wonder if switching from culture media to recording solution might be stressful. Do you give them time to recover?

Are the cells very sensitive to over-trypsinization or overgrowing? Do you have anything in mind when you mention being extra careful?

I really appreciate your help!

I had issues with trypsin while trying to patch dissociated DRG neurons. I would dissociate them in the morning and patch in the afternoon and, if I dissociated for too long, well, no patching. I do agree with Marisol Sampedro Castaneda on the healthy membranes, if the cells look good, they're probably "patchable". And usually Gigaseal issues are indeed related to lack of positive pressure in your pipette. Finally, I am not a super fan of the methanesulfonate internal. It does work, but I'd rather go with gluconate for patching. Did you ever tried a different internal just for the sake of testing?

Thanks Andre Dagostin. Yeah, I've tried gluconate and I didn't notice much, but with the big caveat that I only tried on a few cells from the same coverslip. Do you feel it make a substantial improving the chance of getting GOhm seal? I should perhaps try again, if you feel it makes a big difference.

Interesting about positive pressure. I've only done that with slices. In cultures I advance the pipette until I touch the membrane (based on pipette resistance change), then apply negative pressure.

Hey, Damian J Williams . I have been using gluconate (K and Cs) for the past 4 years now, and it does work nicely, both in slices and culture (HEK cells). Trying it again won't hurt! What I like to do when I face a situation like yours is to remove variables from the equation. If you think your internal is the problem, try different aliquots (maybe one from a buddy in the lab). If no internal works in this specific scenario, the internal is probably not the problem. I will be repetitive now, but pipette pressure is another thing to be really crazy about. Do you see it blowing debris away?

And if after this endeavor nothing improves, I'd check my culture/plating protocols and try to make the cells as happy as they can be.

Is it possible that cells underwent to be ciliated?

Sheng-Nan Wu - thank you! I'm sure that would make it very hard to patch. Is that something which can happen spontaneously? Should I be able to see the cilia under my scope (DIC 40x)?

Andre Dagostin: Just to be sure: do you mean positive pressure and debris for CHO cells? Thanks!

Dissociated or Slice? If dissociated check your enzyme times for dissociations. To much time and the membranes will be very weak and no patch possible as suggested by the other colleagues. To less time to much cellular matrix for a good patch. Next, you have to be sure of the osmolarities of both the extracellular and intracellular solutions. A difference bigger than 25 milliosmols will make again the membrane weak and no patch. If correct, make sure you have positive pressure in the pipette to clean, as much as you can, the cellular matrix around the cell to the best possible. If in a slice, again check the osmolarities and use you positive pressure to clean the cell from the cellular matrix that surrounds it. You'll need more pressure in the slice than in dissociates since there is more matrix that has not been weakened by an enzyme. Good luck!

Thanks all. I am really after tips specifically for patching CHO cells. I'm having no problems with other cell types.

Damian J Williams , that's a general rule. There will always be some debris in the culture/slice which will be blown away by pipette pressure. This is the best way to know if you have good pipette pressure!

Sorry you are still having trouble. The positive pressure and osmolarity are great tips. I use + pressure with any cell type, though less with dissociated cells (about half as much as in slices). Unfortunately I don't have a pressure meter in my current rig but I use a 1ml syringe as a guide to apply pressure, which means it is always more or less the same, and I can adjust depending on the prep. When you are close enough to the cell and resistance changes, releasing this pressure will help a lot with the gigaseal. If the seal is slow, changing the holding potential (depolarizing) gradually also helps.

Looking at your protocol, do you remove the transfection media on day 2 or leave it on until patching day? and do you split the cells before patching or is this not necessary? It could be that cells need to recover from the transfection itself.

Regarding splitting in general, I'm not entirely sure what's best in the case of CHO, but you could try gentler dissociation methods such as TrypLE Express 3-5 min or just a PBS/EDTA 1mM solution for 5 min, then dissociate mechanically.

Hi Damian, Did you try to use the KF based internal solution? I found it very useful for HEK and CHO whole-cell patch-clamp recordings. Here is my recommendation: "Internal solution that contained (in mM): 95 KF, 30 KCl, 1 CaCl2, 1 MgCl2, 11 EGTA, 10 HEPES, 2 K2ATP (pH 7.2 with KOH), fire-polished tips of resistances between 2-5 MΩ. The external (bath) medium contained (in mM) 135 NaCl, 5 KCl, 2 CaCl2, 2 MgCl2 and 5 Hepes, and 10 sucrose (pH 7.4 with NaOH)". The liquid junction potential correction is +7 mV. Pay attention to the Osmolarity difference between solutions,

Good luck