Problem establishing a stable PC12 cell line - (Jun/28/2006 )
I am in the process of establishing a stable PC12 cell line. I would GREATLY appreciate any help/advice and/or protocols that could assist me in this matter. For example, which method of transfections are recommended (i.e. electroporation vs. lipofectamine). Also, what are optimal dish sizes for plating the cells, coating methods, etc. Also, what are optimal G418 selection and maintanence concentrations, and how long should I expect till I can visualize stable colonies The PC12 cells are part of Clontech's TET-OFF cell line. Thank you so much--my initial efforts have failed thus far!
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I am in the process of establishing a stable PC12 cell line. I would GREATLY appreciate any help/advice and/or protocols that could assist me in this matter. For example, which method of transfections are recommended (i.e. electroporation vs. lipofectamine). Also, what are optimal dish sizes for plating the cells, coating methods, etc. Also, what are optimal G418 selection and maintanence concentrations, and how long should I expect till I can visualize stable colonies The PC12 cells are part of Clontech's TET-OFF cell line. Thank you so much--my initial efforts have failed thus far!
max
I have used Effectene fron Qiagen and TransFast from Promega. Both work great! Just follow manufacturer's instructions. I used 6-well plates. Stable colonies were visible within 7-12 days in case of TransFast. I used 3:1 TransFast to DNA ratio. Good luck!
I am in the process of establishing a stable PC12 cell line. I would GREATLY appreciate any help/advice and/or protocols that could assist me in this matter. For example, which method of transfections are recommended (i.e. electroporation vs. lipofectamine). Also, what are optimal dish sizes for plating the cells, coating methods, etc. Also, what are optimal G418 selection and maintanence concentrations, and how long should I expect till I can visualize stable colonies The PC12 cells are part of Clontech's TET-OFF cell line. Thank you so much--my initial efforts have failed thus far!
max
I have used Effectene fron Qiagen and TransFast from Promega. Both work great! Just follow manufacturer's instructions. I used 6-well plates. Stable colonies were visible within 7-12 days in case of TransFast. I used 3:1 TransFast to DNA ratio. Good luck!
Great, thank you for the advice! BTW, any luck with Lipofectamine because its sitting in my fridge?
I'm also in the process of trying to make a stable PC12 cell line. I can say from experience that these cells are notoriously difficult to transfect with high efficiency using common lipid-based reagents. While Lipofectamine 2000 does transfect PC12 cells, it is of low efficiency and not suitable for all assays (especially siRNA in my hands). I would say that high transfection efficiency is very important, as it increases the likelihood of a stable recombination event within your cell population.
Here's a good paper describing optimized conditions for PC12 electroporation, which I've done with some success:
Ghosh C, Song W, Lahiri DK. Efficient DNA transfection in neuronal and astrocytic cell lines. Mol Biol Rep. 2000 Jun;27(2):113-21.
Here's a good paper describing optimized conditions for PC12 electroporation, which I've done with some success:
Ghosh C, Song W, Lahiri DK. Efficient DNA transfection in neuronal and astrocytic cell lines. Mol Biol Rep. 2000 Jun;27(2):113-21.
Thanks for the advice. I agree, I have considered using the Amaxa electroporation system for future transfection of my PC12 cells. Are you electroporating your PC12 cells, and then plating them in a 24 or 48 well format, using poly-d lysine or collagen as a substrate?