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
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
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.
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.
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