internal solution-Patch clamp-hERG assay

5 posts / 0 new
Last post
Sebastian Joseph
Sebastian Joseph's picture
internal solution-Patch clamp-hERG assay

I am using hERG HEK cells for drug screening using automated electrophysiology system. Could anybody clarify/comment on following points
1. For experiment I harvest 3 day old cells (70% confluency) using Detachin (1ml per T75 flask for 3 - 5 min). Cells are going in to whole cell mode at very low pressure i.e  -80 to -120mB and it appears that they are very fragile. For pasaging I reduced my trypsin strength from 0.05% to 0.01 % for 3 min, still the problem persists. Can anybody suggest better option to make the cells more strong?
2. These cells form gigaseal but seal remains for few minutes only (cell looses seal after few minutes and recording can not be continued). Could this problem related to my solutions or cells? What changes in the internal solution we can make to get stable cells?
3. I am experiencing some difficulty in geting a clear internal solution. When I add mgcl2 the solutions turns white and it will not become clear even after PH adjustments. Can you suggest ways to prepare internal bath solution? My internal solution contains EGTA, mgcl2, Na-ATP, kcl, kF, HEPES. I adjust the pH using KOH
 

zhangxu04
zhangxu04's picture
I do the same experiment

I do the same experiment using AXON 200B
my cll was not only fragile but aslo very  viscidity and is very slow to fomr the G seal. so I reduce the opening of the pipette.
so I think it will be helpful to do some change to your pipette

Bluejay
Bluejay's picture
Which automated system are

Which automated system are you using?  Some people have problem with HEK cells sealing to plastic patch plates.
People have problems with grape-like clusters of HEK cells.  These can wash off some systems or start as a multicell clamp and then break off. 
My impression is that HEK expression causes cell swelling, so perhaps a lower expression level cell would help.  You could centrifuge at a lesser speed or shorter time.
Generally a white precipitation in these buffers is due to calcum phosphate but perhaps this is not your issue since you are using a HEPES buffer.
You can try replacing 40mm of internal KCl with 40mM KF, which stabilizes some cells.
You can try Versene to detach the cells.   Also, in my case, cells were very sensitive to placement in Versene at 37C.  So perhaps you could use reduced temperature even though it takes 20min. or so to detach the cells.
Hope some of this helps!

Fraser Moss
Fraser Moss's picture
Normal

Normal
0

false
false
false

EN-US
X-NONE
X-NONE

MicrosoftInternetExplorer4

/* Style Definitions */
table.MsoNormalTable
{mso-style-name:"Table Normal";
mso-tstyle-rowband-size:0;
mso-tstyle-colband-size:0;
mso-style-noshow:yes;
mso-style-priority:99;
mso-style-qformat:yes;
mso-style-parent:"";
mso-padding-alt:0in 5.4pt 0in 5.4pt;
mso-para-margin:0in;
mso-para-margin-bottom:.0001pt;
mso-pagination:widow-orphan;
font-size:11.0pt;
font-family:"Calibri","sans-serif";
mso-ascii-font-family:Calibri;
mso-ascii-theme-font:minor-latin;
mso-fareast-font-family:"Times New Roman";
mso-fareast-theme-font:minor-fareast;
mso-hansi-font-family:Calibri;
mso-hansi-theme-font:minor-latin;
mso-bidi-font-family:"Times New Roman";
mso-bidi-theme-font:minor-bidi;}

For dissociating the HEK cells, you can try cell stripper liquid from Mediatech. It's a non-enzymatic cell dissociation solution.

http://www.cellgro.com/shop/customer/home.php?cat=358

An Alternative is made by sigma

C5789 Sigma Cell Dissociation Solution Non-enzymatic 1X

http://www.sigmaaldrich.com/catalog/search/ProductDetail?ProdNo=C5789&Brand=SIGMA
 
HEK's are not very sticky so you could also manualy bash the flasks and titrurate them to break up cell clumps
Regarding the sealing problems - do you check the osmolarity of all your solutions?  First compare the osmolarity of your solutoins compared to the culture media in which they grew.  You don't want then to suffer osmotic shock when you transfer then to the high throughput patch device.  Culture media and recording solutions typically have osmolarities of 290-300 mOsm/L.  Also you should make the intracellular "pipette" solution slightly (~5%) hypotonic compared to the extracellular solution.  In conventional patch this improves the seals because the stream of internal solution bathing the cell wehn you approach to patch causes the cell to swell slightly and forma quick Giga seal.
 
here is my internal solution for hERG - I have never had problems with precipitation of any of the components

Normal
0

false
false
false

EN-US
X-NONE
X-NONE

MicrosoftInternetExplorer4

/* Style Definitions */
table.MsoNormalTable
{mso-style-name:"Table Normal";
mso-tstyle-rowband-size:0;
mso-tstyle-colband-size:0;
mso-style-noshow:yes;
mso-style-priority:99;
mso-style-qformat:yes;
mso-style-parent:"";
mso-padding-alt:0in 5.4pt 0in 5.4pt;
mso-para-margin:0in;
mso-para-margin-bottom:.0001pt;
mso-pagination:widow-orphan;
font-size:11.0pt;
font-family:"Calibri","sans-serif";
mso-ascii-font-family:Calibri;
mso-ascii-theme-font:minor-latin;
mso-fareast-font-family:"Times New Roman";
mso-fareast-theme-font:minor-fareast;
mso-hansi-font-family:Calibri;
mso-hansi-theme-font:minor-latin;
mso-bidi-font-family:"Times New Roman";
mso-bidi-theme-font:minor-bidi;}

Internal Pipette solution

mM     Compound        

 
110     K-gluconate          
20        KCl                          
1          MgCl2.6H2O         
5          EGTA                  
5          MgATP                      
10        HEPES                     
 
pH to 7.4 with KOH. Expected about 280 mOsm/kg
 

Fexofen
Fexofen's picture
I think the precipitate you

I think the precipitate you are seeing is a salt of MgF which is very insoluble in water. In Fluoride containing internal solutions avoid using Mg+2 or Ca+2 as their salts with F are very water insoluble. For hERG recordings there is no need to use KF in your solutions. See if you can reduce the suction pressure while forming whole seal (if cells go into whole cell quickly) and increase the time given to form giga seal. Also if you are losing cells quickly during the experiments try increasing the holding suction and make sure that compound addition does not cuase you to lose cells due to disturbance while addition.
Hope this helps