Ok guys

I've never been on this site its te first time i've discovered it and it seems pretty useful as I've been reading different threads and posts on here.

I've been using the HT ionworks machine and have been experiencing problems with it

No matter how careful we are with cells were still getting large seal failures ...

1) can it be due to low channel expression or vaccume under the patchplate?

2) can the passage number of cells have and affect on cell size and therefore leading to large number of seal failures?

3) anyone ever looked into how the cell size can affect the seals of cells on a patchplate?

4) the antibiotic used for selecting the whole cells that express the channels ... is there any test to determine if the cells have developed a resistance to the agent and therefore no longer selective?


sorry for asking too many questions ... is anyone using the same machine? its been a pain getting this thing to optomise

we get odd days when the assay has worked really well and then a few odd weeks where its no longer functioning anymore ...

That was *Need 'Some' answers A.S.A.P :-)

I precede these answers with the statement that I personally have not used the ionworks HT but am very familiar with the machine and work in a lab the specializes in conventional patch electrophysiology

I doubt your problem it is due to low channel expression. In general I have found non-transfected cells usually give the high seal resistances. If you want to check your channel expression levels, just pop a few on a conventional rig and do some whole cell experiments.

What cell lines are you using and what channels are you expressing?

How are you dissociating the cell from culture flask and putting them in solution for patch? You will want to avoid trypsinization and use at most an enzyme free dissociation cocktail, or just mechanically dissociate the cells with a cell scraper or bashing a flask to preserve membrane integrity. HEK293 cells in particular do not like enzymatic dissociation.

In general, try to avoid using cells more than 15 passages after thawing because their morphology (and cell size) can change dramatically after that. Furthermore if you are using a stably expressing line you should be careful that the cell line does not "spit out" the stably transfected channel or one of its subunits after 12 or so passages (Ive had this happen with calcium channel subunits while the cell still retains the antibiotic resistance cassette).

To improve seal quality you can make your intracellular solution have a lower osmolarity compared to the extracellular solution by about 10%. This encourages the cell to swell slightly and form a tighter seal with the chip before you perforate. Additionally you should try and match the osmolarity of your extracellular solution to the culture media in which the cell grew so that they do not swell or shrink too much during the preparation for patching.

Do you use amphotericin free intracellular solution while you attain your seals and then switch to solution with the perforant? If you have amphotercin in the intracellular solution while you are trying to get your seal it may be that you are not forming your seal fast enough and amphotericin is getting into both intracellular and extracellular solutions and completely perforating many of your cells resulting in extremely poor seals.

You may also want to read some of the technical tips from the Molecular Devices web site:

Eg. Increased seal resistance by placing Patch Plates in a dessicator
http://www.moleculardevices.com/pdfs/IonWorks_Technical_Tip_1.pdf

Other technical tips are at
http://www.moleculardevices.com/product_literature/family_links.php?familyid=10


I hope Ive been some help. Let us know how you fixed you problem even if it is not any of my suggestions because it will be useful for other people in a similar position to yourself. Good luck.

Hello Fraser

Thanks for replying back really appreciated your advice and feedback on some of the issues we're experiencing.

Im using Chinese Hamster Ovary cells for Herg channel expression.

The cells are dissociated in D-PBS (+ cacl2 + mgcl2 ions) . I've been avoiding the trysinizing step as it affected the cell fragility and have replaced it with a lighter agent - Versene.

I'm investigating further in to the passage numbers and how it affects our overall success rate as the cells age and change in morphology.

Osmolarity is something I've not looked in to yet but thats definitely a good point! (I'm sure when the machine was validated these thing were taken into consideration but since then Ive really not had a look and see whether this is something thats effecting the cell performance)

We use intracellular solution first for the cells to form seals and then have amphotericin which circulates through the plenum however we have adjusted the seal delay time a few times to see if it improves seals but have had no success.

Its been an on going process the minute it starts working we think theres hope but then something else blows up on the machine which leads us to start all over again and start investigating into what the next problem is

Another thing which Ive heard is the patch plates coating, the plates we get are not QC checked for resin coating. The resin on the patch plates is meant to help the cells to adhere so yes any of my ionworks friends who are using these patch plates and are experiencing problems like myself thats something to definitely keep your eyes on

FYI: do you sonicate your amphotericin immediately before you perfuse it through? This should be a big help it attaining perforated patches "quickly" while your seal quality is still good.

Hello again

yes we do normally for around 30minutes just before experiment

Dear Lost Marbles,
I have put some suggestions to your questions

Can it be due to low channel expression?

Why not put the cells on your conventional rig and manually patch to look at expression levels, about 10 cells and you should have an idea of the expression level in the cell population.

There are lots of tricks for increasing channel expression, my favourite is using the 28oC method for a few days before patching.

Or why not try some transient transfections?

Can the passage number of cells have and affect on cell size?

When you set up the cell line did you take pictures to see if cell size changed over time. When we validate a cell line we take pictures/measurements that can be compared over time for just this type of trouble busting.

The antibiotic used for selecting the whole cells that express the channels ... is there any test to determine if the cells have developed a resistance to the agent and therefore no longer selective?

Again, check your expression level of channel if there is reduced antibotic selection, you will have a population of cells that do not express channel ie wild types.

we get odd days when the assay has worked really well and then a few odd weeks where its no longer functioning anymore ...

If your results are eratic this can usually suggest that the cells aren't happy. Has something changed in your lab?

As mentioned above changes in osmolarity can be a problem. Also, measure the osmolarity of our solutions, don't rely on theoretical calculation of it, as in my experience the actual value can often be lower than the theoretical value. If different people are weighing stuff out when making the solutons then the osmolarity can change day to day.

Could your cells be sensitive to temperature, are you pre-warming your dissociation solutions so you don't cold shock them?

Hope this helps.

Am having similar problems getting hERG CHO cells to work on Ionworks. Web presentation from Roche and talks from Essen suggest just trying 10-30 different clones. Lower seal cutoff is 50mOhm but many researchers get about 100-200mOhm. Am interested in how people are progressing on this troubleshooting. In our case, I don't think it's the cells. Gentler/shorter Versene exposure may increase the seal rate. Haven't found a good practical guide for planar patch clamp since it's a relatively new technique.