Which transfection reagent was used? I'm having a real hard time transfecting it.
Hi Mor Abutbul, try the amaxa Nucleofector: http://www.lonza.com/products-services/bio-research/transfection.aspx. Contact their scientific Support or check the Cells & Transfection database. There are transfection protocols using the Nucleofector Technology. Good luck. Bodo
I used to transfect the cell line before. I used the Turbofect from ThermoScientific as the transfection reagent . Although the efficiency was not so high. See the link for the detail:
http://www.thermoscientificbio.com/transfection/turbofect-transfection-reagent/
Hi Mor, we have done this and used the resultant cells to make clones from the transfection. We also used Turbofect. I have included the protocol below.
We did this quite a while ago (and my memory is not great) but if I remember the % transfection was not great (~10-20%) so you may want to generate stables.
Note: H9C2 cells can methylate some viral promoters and you will lose fluorescence of stable transfectants over time (esp. CMV promoters: see:
"Effects of epigenetic modulation on reporter gene expression: implications for stem cell imaging" Krishnan et al 2005, The FASEB Journal).
CELL TRANSFECTION IN HEK-293 and H9c2 (2-1) cell lines
In each well of a 6-well plate, seed either HEK-293 or H9c2 (2-1) 5x105 cells in 4ml of complete growth media one day before transfection. Cells have to be between 70-90% confluence on the day of the transfection.
Dilute 2.5ug of pVisionGFP-C Vector (BioVision; Cat no# 9998-20) or pVisionRFP-C Vector (BioVision; Cat no# 9996-20) in 250ul of DMEM or any other serum-free growth media into a 1.5ml microfuge tube.
Briefly vortex the TuboFect transfection reagent and add 6ul of it to the diluted DNA sample. Mix immediately by pipetting or vortexing.
Incubate the samples at room temperature for 20 minutes.
Add 250ul of the Turbofect/DNA mixture to the appropriate wells on the 6-well plate, making sure not to remove the growth media from the cells (3.75mls/well).
Gently rock the plate to achieve an even distribution of the TurboFect/DNA complexes in the wells.
Incubate the plate in a 37oC/5%CO2 incubator.
Transgene expression can be seen under fluorescence from around 6 hours post-transfection.
For stable transfection, cells should be grown in selective medium containing 400ug/ml G418 Disulfate Salt Solution (Sigma; Cat no# G8168) from 48h post-transfection up to a total period of 10-15 days.
Hope this helps,
Gary
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