I would like to use Lipofectamine 3000 (Life technologies) to transfect human embryonic stem cells (H9) and make the selection by sorting with FACS aria II. My basic concerns includes -
Whether, I should be having my normal culture media (i.e mTeSR) or Opti-MEM (reduced serum) while transfecting the cells in culture?
Also, does it make any difference, if changing media after 6-16 hrs post transfection with reagent+DNA free media?
How longer I, should culture my transfected cells before going for selection process with FACS (eg. 24 hrs, 48hrs)?
Many thanks for your advise and suggestions in advance, Reena
Hi Reena
1) I am not really familiar with lipofectamine 3000, but a colleague is performing transient transfection using his pluripotent medium (in this case iPS Brew from Miltenyi) with acceptable results.
2) Would be better to change the medium, even if your transfecting agent should shows less toxicity compared to other agents.
3) That's tricky because mainly depends from the type of vector you are introducing in your cells: assuming that you have a GFP reporter under a constitutively active promoter, I think that 24 hours is not too early for sorting. In any case I suggest you to compare the two time points and verify if you observe a drop in the GFP (or other marker) intensity.
Good luck with your experiments!
Alessandro
Dear Reena,
For transfecting your H9 cells, my recommendation would be to use a specific reagent for stem cell transfection such as DreamFect Stem
http://www.ozbiosciences.com/transfection-dna/17-dreamfect-stem-transfection-reagent.html
Regarding the culture medium, I suggest using OptiMEM to prepare you DNA/transfection reagent complexes and then your normal culture media for culturing your cells
Sometimes yes, changing the culture medium after 4 hours for example, does affect the cell viability, so if your cells are very sensitive to transfection, this can be a good option.
For selection process, generally 48 or even 72h is good before applying selection
Good Luck
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