Hi all,
I am about to perform Lipofecatmine 2000 kit-based transfection in SHSY5Y (mammalian) cells. I have some questions regarding the minute details in the protocol, which I hope you can help with!
So for example if I am using a 24w plate, it is said to seed the cells in 500uL of plating medium.
1. I understand presence of FBS in media can affect the transfection process. For this reason, I had tried to wean my cells (which were originally growing at 10% FBS) to 1% FBS. So from 5 to 2 to 1%. The moment I remove serum completely from the media, the cells do not survive. Would it still be ok to plate the cells in 1% FBS for transfection?
2. I understand Opti MEM is a reduced serum formula recommended to use with Lipofectamine 2000. However, this media is pretty expensive and currently I have only gotten 50mL of it. Based on the protocol, it seems as if we only need to mix the DNA and Lipofecatmine complexes in Optimem and we can add this directly to the cells. So is it correct of me to think that we do not need to plate all the cells in Optimem (ie. can plate with my own DMEM and just add the DNA-lipid complexes diluted in Optimem to the wells?)
3. If Optimem is a reduced serum formula, should I add low percentage of serum to it, or can I use it as it is?
Thank you in advance and I really hope someone can help me with these queries before I start my experiment next week!!
Best Regards,
Mathangi
Hi Mathangi Palanivel
L2K has the a low transfection efficiency with a high toxicity on SH-SY5Y cell line.
FBS has a different effects on the transfection efficiency, sometime 20% FBS achieved the highest transfection efficiency and in others 0%. In case of SH-SY5Y cell line with L2K 10%FBS is a good choice.
Different cell culture media can be used in the case of SH-SY5Y such as DMEM and 45% EMEM supplemented with 45% Ham’s F-10. You don't have to use Opti-MEM as a culture media and it is recommended only for incubating the nucleic acid of interest with the transfection reagent.
Of note, each lab has its own recommendation regarding transfection, you also should to optimize your ones to get the highest percentage of transfection efficiency according to your experiment conditions.
All the best.
Hello Mathangi Palanivel
1. Would it still be ok to plate the cells in 1% FBS for transfection?
Yes. Did you try 0.5% serum? Do your cells survive in culture media supplemented with 0.5% serum? Serum free or serum‐reduced media is recommended for transfection simply to synchronize the cell cycle and reduction of serum protein interference during transport of genetic material into the cells. So, use 0.5% serum, if your cells cannot survive 0.5% serum, then you may use 1% serum.
2. So is it correct of me to think that we do not need to plate all the cells in Optimem (ie. can plate with my own DMEM and just add the DNA-lipid complexes diluted in Optimem to the wells?
Yes, you are right in your thinking. Opti-MEM is used to dilute Lipofectamine and DNA before complexing. You can plate your cells in 0.5% or 1% serum containing media and then DNA-Lipofectamine 2000 complexes diluted in OptiMEM can be added directly to cells in culture media.
3. If Optimem is a reduced serum formula, should I add low percentage of serum to it, or can I use it as it is?
No need. OptiMEM can be used as is. Other culture media (e.g. DMEM) may be used without serum to dilute Lipofectamine 2000 and DNA, but transfection efficiency could be compromised. Since you have 50ml OptiMEM, I suggest you use OptiMEM for diluting DNA and Lipofectamine2000 for better results.
Good Luck!
Malcolm Nobre Mohamed Khashan thsnk you so much for your very valuable inputs!
Hi! I'll do my best to answer your questions:
I hope that helps! Good luck with your experiment.
Ahmad Al Khraisat Ok thank you so much for the clarification, especially the use of OptiMEM for the dilution of complexes!
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