I am trying to transfect HEK293 using MegaTran with l type subunits but I was not successful. Does anybody have advice relating to this protocol? Another problem is aggregated cells. What is the best was to prevent the aggregation of cells?
You can find the cell culture protocol at ATCC attached below.
http://www.atcc.org/products/all/CRL-1573.3.aspx#7301B7F956944F8382B6192957C08A3B
For patch-clamp recording,
You can try the following protocol and bath/pipette solutions:
the voltage step (from -60 mV to 80 mV by 10mV) with 300 ms duration, and cells were maintained at a -90 mV holding potential during experiments.
Pipette Solution: 115 mM Cs-methanesulfonate, 10 mM Cs-1,2-bis-(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (Cs-BAPTA), 5 mM CaCl2, 8 mM MgCl2, and 10 mM HEPES (pH adjusted to 7.2 with CsOH). Bath solution: 120 mM Tetraethylammonium chloride, 10 CsCl, 10 mM CaCl2, 10 mM HEPES (pH adjusted to 7.4 with CsOH).
Thank you Xuexin for your input was very helpful , i am facing the issue of unhealthy cells due to transfection reagent and difficulty in patching since they are moving
I've used Effectene Transfection kit from Qiagen with calcium channel and HEK293 cells in the past with great success. They were healthy enough to patch too. I'm not familiar with MegaTran protocol though. Good luck!
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