Anyone got a good cure to stop access resistance creeping higher?

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Fraser Moss
Fraser Moss's picture
Anyone got a good cure to stop access resistance creeping higher?

I'm doing some dose responses on mammalian cells that take over 1 hour to get all my concentrations because of slow onset of drug effect.

During that time my access resistance of my Whole cell seal is creeping up from approx 10 MOhm to 30 -40Mohm.

Anyone seen this happen much and got any good remedies?

FYI my pipette soln is

mM Compound

110 Kgluconate
20 KCl
1 MgCl2.6H2O
5 EGTA
5 MgATP
10 HEPES

littlebush
littlebush's picture
I guess you used the

I guess you used the conventional ruptured whole cell recording,right?If so,the most possible reason for your increasing series resistance is due to the resealing of the ruptured membrane,and I strongly suggest you use the perforated patch clamp technique to do it,especially the amphotericin B as the perforating agent because of reported lower access resistance than other perforating drugs.

The perforated patch clamp is not difficult to perform and the most troublesome step may lie in the preparision of the amphotericin B which is insoluble in water.The follow review is very good for knowing nearly everthing concerning the perforated patch clamp ,Nystatin perforated patch recording and its applications to analyses of intracellular mechanisms.
Finally,I want to stress that the perforated patch clamp is extremely stable for a long time recording which seems particularly suitable for your needs.

Good luck and happy patching.

Fraser Moss
Fraser Moss's picture
Thank you

Thank you

I was aware of the perforated patch option but because I have so much data already with ruptures whole cell I was hoping to finis this study without switching techniques.....looks liike I might have to though

littlebush
littlebush's picture
Hi,frasermoss.Yeah,it is well

Hi,frasermoss.Yeah,it is well known that the resealing of the ruptured membrane is a chronic headache for the conventional whole cell recording during long time experiment.Maybe,the reasons underlying this is not well characterized but it has been pointed out by some authors that the presence of relatively high Ca2+ concentration around the membrane will facilitated this resealing process.Accordingly,so ,maybe it is advisable to enhance the EGTA concentration .I do not know your specific experiment scene but you may have a try.In addtion ,I want to know the resistance of your pipet and it is better to use lower resistance pipet as possible aiming at enlarging the pipet's tip size given such adjustment not interfering with tight sealing.
Best wishes with your experiment.

littlebush
littlebush's picture
Hi,frasermoss.I think that

Hi,frasermoss.I think that you should provide much more detailed information about your specific experiment,say,voltage clamp or current clamp?what's your objective?what's the ion channels you are interested in?Is that voltage gated or ligand operated channels?Will the creeping access resistance do harm or not,just depends on what the specific experiment condition.The main drawback of the seriess resistance is that it will cause pipette voltage drop which in turn can result in erroneous command voltage on the cell,but if the current is not large say calcium currents then such increased access resistance will be not of big problem or you can correct it by calculation.

Good Luck.

Fraser Moss
Fraser Moss's picture
Whole cell voltage clamp

Whole cell voltage clamp

CHO cells

hERG potassium channels

Dose Response experiments to hERG blocker drugs

It seems that access resistance does creep sometime by as much at 20Mohm over the course of the expt. I can fix that with extra compensation during the experiment.

My main problem is now channel rundown and deteriorating membrane resistance (increased leak current) over the 80 mins it takes to get a full DR of a cell with my protocol (it takes 10 mins for each drug to equilibrate because they cross the memebrane and block from the inside of the pore). I do have Mg-ATP in my pipet, but am thinking of adding cAMP and/or PIP2 as well which are known to modulate hERG and potentially prevent rundown.

I have tried perforated patch, but it is an enormous pain in the posterior, waiting for perforation to occur and then it often just blows the cell (used 0.24mg.ml Amphotericin B).

I'd love any feedback from any hERG experts out there who have done lots of dose responses on cells patched in the whole cell voltage clamp mode on mammalian cell lines expressing hERG and have negotiated the run-down issue.

littlebush
littlebush's picture
Since you believe your drug

Since you believe your drug acts on the inside of the channles mouth and if you set up is suitable for low noise recording,why not trying the cell free inside -out mode to perform your experiment,which is much more informative in investigating the channels drug interaction.

Golden ball
Golden ball's picture
I have a primitive question.

I have a primitive question. You keep your whole cell patch for more than 1 hour. What is the seal value at the beginning and the end? 3G and 2G? or 3G and 200MOhm? 3G and 10G (resealing?)? Myself, it is very diffciult to keep an acceptable whole cell configuration for such a long period.

Fraser Moss
Fraser Moss's picture
yes the update is that the

yes the update is that the problem is deteriorating seal resistance and that I am recording for shorter times and taking less data points (drug concentrations) per cell.

Going to retry perforated patch again too.

littlebush
littlebush's picture
Air polishing your pipette

Air polishing your pipette and keeping your solution both bath and pipette very clean are of paramount importance to obtain high and long standing seal which obviously will aid the long standing recording.However,as for the access resistance in the ruputured whole cell mode,it is an inborn headache for the increasing access resistance,or to be more precise,it is inevitable during the conventional whole cell configuration.The detrimental extent depends on both the size of this access resistance and the current size of your cell,the error can be calculated by just Ohm's law.One remedy is to check the access resistane every several mins and re-compensate it but another way,also in my opinion the best way ,is to use perforated patch technique .The rational lies in that after reaching stable partition .the access due to the partioning hole in the patch membrane is very stable and can last at least 3 hours which is the active period of the antibody in the solution.In short,perforated patch is the best and it is really worthy of tring.In addtion,if your drug channel interaction involve with intracellular singling pathway,it certainly add invaluable weight.

Golden ball
Golden ball's picture
Hi,

Hi,
I attached an article regarding perforated patch using nistatin. HOpe you would find usful infomation. About hERG assay, do you drive the cell with the voltage step protocol from -80 mV to +20~30 mV? I put a short voltage step at -50 between the holding potential at -80 mV and +30 mV. And I measure the tail current betwewen the current at -50 mV and the peak value of the tail. This can reduce somehow the distubance from the leak, since the current at -50 mV seems to be closer to the baseline as far as the leak is not such a huge. This insert of voltage step at -50 mV for 200 msec helps me for hERG assay.

Fraser Moss
Fraser Moss's picture
Thanks for al the tips...I'm

Thanks for al the tips...I'm actually alread implementing 90% of them already. Especially the polising of pipettes, filtering of solutions and sampling of Ra and Rs during the protocol.

I think when I retry perforated patch I'm going to try Gramacidin because the pores it creates don't have any Cl- conductance.

By the way, it's great to have some other electrophysiologists on the site now. I hope you all continue to post questions and answers and we can form a healthly sub-community here.

littlebush
littlebush's picture
Though,the Gramacidin sounds

Though,the Gramacidin sounds the best because it is inpermeable to Cl .However,the partitioning time is the longest so if your experiment do not focus on the Cl channel.I do not think it is your option.Good luck