pipette solution for HEK293 whole cell recording

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HY
HY's picture
pipette solution for HEK293 whole cell recording

Hi,

I am doing whole-cell recording on HEK293 cells. I found that my control group, eGFP-transfected cells, gave me variable whole cell currents which range from 50 pA to 500 pA. Those cells look very healthy. I tried to optimize the internal solution. When I read literatures, there are many different protocols. Could anyone give me a good suggestion? 

Thank you. 

HY 

The FFM
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What channel type are you

What channel type are you trying to record ?

The type of currents that you are trying to record dictate the composition of the ECS and pipete solution

HY
HY's picture
Hi,

Hi,
 
I am recording hemichannels. my pipete solution is Cscl 130, EGTA 10, CaCl2 0.5, Hepes 10. Extracellular solutions: NaCl 140, CsCl 2, CaCl2 2, MyCl2, KCl 4, Hepes 5, glucose 5, I am wondering why control groups are so variable. Do you have good suggestions? Thanks.

egovoru
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 Do you use transient

 Do you use transient transfection or a stable line? If the former, then the high variability in current amplitudes is quite normal, most likely because of variable expression levels in individual cells. 

green_bear
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 Hi all,

 Hi all,

I am trying to find a pipette solution with ion composition most resembling to the intracellular ion composition of HEK293 cell. I am not trying to measure a certain ion channel, but rather to test how the transfected channels would function in the cell in combination with other (endogenous) ion channels. 
I would appreciate any suggestion on this. 
Also, if anyone has a good reference for guide on choosing pipette solution in whole-cell patch clamp, that would be fantastic.

Great thanks,
g_b