sealing problems : HEK293

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zhangxu04
zhangxu04's picture
sealing problems : HEK293

I have made patch clamp on HEH293 more than 2 month. but  I could not sealing problems the sealing problems.
the sealing could lncrease only 40-50M amlost no G seal.
could somebody give suggsetion ?

Fraser Moss
Fraser Moss's picture
If you replated how long did

If you replated how long did you wait between repating and patching and what did you use to dissociate the cells before you replated (Trypsin/enzyme free dissociation solution/manual dissociation [scraping/lifting]?)
Have you checked the osmolarity of your external and pipette solutions?
Check the plastic pipette holder for cracks and the rubber seals inside for wear and tear in addition to all your tubing.
How big is your internal tip diameter?  if it is too big it will be hard to hold a giga seal for long and if it is too small they may clog or reaseal.  1-1.5 µm is usually ok.

zhangxu04
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to frasermoss :

to frasermoss :
patch clamp  is a so difficult work for me. please help me!!!!
I use only Trypsin to dissociate the cells with manual lifting. than I will make patch clamp after 24-36 hours.  my  osmolarity of your external and pipette solutions are just ok.  I have no way to measure the opening of the tip. but usually the resistence  is about 3-5M ohm, now I increas about 7-8Mohm when just in bath.  could you give me some suggestions?
I think my cell is not very helthy. because there is much little spot on the surfance of cell.
when touched the cell and the resistance increas about 0.2-0.3M ohm, I suck the pipette, then the resisitance increas about 40 or 50Mohm, then the resisitance stop increas and beging decease about 10-20Mohm. I guess the seal is leaky. could you explain how the seal become leaky ? I remember  you have said if the tip was too small, it was very possible to cause the seal leak. I d not know how the leak happen. my cell seems very delicate, so this may be prone to be leaky.

Bluejay
Bluejay's picture
Suggestion:

Suggestion:
Try different detachment buffers such as Versene or Sigma's cell dissociation buffer.
Minimize the time cells are exposed to trypsin, minimize trauma due to lifting the cells off the plastic, and minimize the centrifuge  force and the time cells are sitting as a pellet.  Of course, trypsin effects must be stopped by adding media with serum.  Use  trypan blue to assess cell viability.
Cell handling is an art.  Each cell line is a little different and may require slightly different handling techniques. 
Corning has very useful cell handling webinars online.

Fraser Moss
Fraser Moss's picture
zhangxu04 wrote:

zhangxu04 wrote:

to frasermoss :
patch clamp  is a so difficult work for me. please help me!!!!
I use only Trypsin to dissociate the cells with manual lifting. than I will make patch clamp after 24-36 hours.  my  osmolarity of your external and pipette solutions are just ok.  I have no way to measure the opening of the tip. but usually the resistence  is about 3-5M ohm, now I increas about 7-8Mohm when just in bath.  could you give me some suggestions?
I think my cell is not very helthy. because there is much little spot on the surfance of cell.
when touched the cell and the resistance increas about 0.2-0.3M ohm, I suck the pipette, then the resisitance increas about 40 or 50Mohm, then the resisitance stop increas and beging decease about 10-20Mohm. I guess the seal is leaky. could you explain how the seal become leaky ? I remember  you have said if the tip was too small, it was very possible to cause the seal leak. I d not know how the leak happen. my cell seems very delicate, so this may be prone to be leaky.

 
First - FYI The URL for those corning webinars mentioned by Bluejay is http://www.corning.com/lifesciences/emea/en/technical_resources/online_training.aspx

 
In reply to point 1.  As Blujay said, avoid trypsin use if you can.  In addition to lifting the cells trypsin will also digest any ion channels at the surface of the cells when you do the dissociation so you will have to wait 24h or more for the expresssion to normalize. HEK293s are not very adherant and you can dissociate them easily by manual bashing ofthe flask or by changing the nedia in a dish (3.5cm) , rinsing and then adding 1ml of the ECF that you will you to do recording.  Let it sit for 15 mins and then using a 1000µl pipetteman titurate the cells off the dish and replate If you must use a dissociatoin agent as BlueJay said use a non-enzymatic agent like cellstripper from cellgro, or Sigma's cell dissociation buffer.
RE the tip size.  Ask around the department to see if anyone else owns a micriforge. Or make your own arrangement with a binocular dissection microscope and a spare pipette holder.  Place a graticle in the eypiece and calibrate the size of each division using a known measure.  Then place the tips below te scope and measure the tip diameter.  You will also be able to see the shape.  You might be pulling a 3MOhm electrode but the tip might be an aweful shape.  Some aplications require that you also fire polish the tips (using the microforge) to get better seals.  If your tips are too small you will apply too great a pressure at the membrane below the tip and rupture it too volently often before you get a Gigaseal and end up with a leaky seal.
Your experince trying to get a seal sounds a lot like you have a leak somwhere between the suction and the pipette tip.  Have you changed all you tbing and rubber seals in the pipette holder yet?  Do it.  Sometimes you just cannot see or hear a leak but changing the tubing can make all the difference.  Also change the valve that you are using to lock the suction once you apply suction.

zhangxu04
zhangxu04's picture
thank you very much. I

thank you very much. I appreciate all the suggestion you guys gave me.
 

zhangxu04
zhangxu04's picture
to frasermoss :    how to

to frasermoss :
   how to dissociate them easily by manual ?  I grasp your meaning that I first rinse my cell in dish with media sereval times and the add 1ml ECF (what ECF stands for ?). is it that I have to first throw away the media from  dish and then add ECF or  add the ECF into the media?

Fraser Moss
Fraser Moss's picture
suck off the culture media

suck off the culture media
wash quickly in 1ml of Extracellular Fluid (ECF; the solution you use to bathe your cells when you are patching)
Add 1ml of ECF and let them sit in it for 15 min.
Then using a pipetteman and a 1ml tip just gently suck the ECF up and down and blast the cells (not too hard!) off the bottom of the dish.