Usually neuroblastoma have low transfection efficacy (5-10%) when transfected with Lipofectamine 3000. So, this time I have tried electroporation with this condition,
poring pulse: 175V, 2.5ms, 50ms interval, 2 pulses, 10% decay rate, + polarity
transfer pulse: 20V, 50ms, 50ms interval, 5 pulses, 40% decay rate, +/- polarity
I have used this transfection condition with three seeding density (80K, 100K and 150K)in 24 well plate.
However, the cell viability is very low for RNA collection after 24 hour of electroporation.
Could anyone please suggest me how to improve the cell viability?
TIA.
Regards
Ayman
Dear Ummay Ayman
Optimizing electroporation conditions for transfecting SH-SY5Y cells involves a series of experimental steps to determine the best parameters for your specific application.
Voltage: Start with a lower voltage (e.g., 100-300 V) and gradually increase until you find the highest voltage that gives acceptable cell viability and transfection efficiency.
Pulse Length: Generally, shorter pulse lengths (milliseconds) are preferable. Start with a range of 1-10 ms.
Number of Pulses: Try a single pulse initially and then test multiple pulses if needed.
Pulse Interval: Depending on your cell type and the pulse parameters, you might need to adjust the interval between pulses.
Kind regards
AB Bayazid
https://www.nature.com/articles/s41598-021-94620-8
Dear A.B Bayazid
Thank you for your reply.
The mentioned poring and transfer pulse conditions are used in my lab after optimization in case of SHSY5Y.
However, I was not getting enough cells may be because of technical errors as I am doing this first time.
So, following some literature protocols, after electroporation, I have seeded 1M cells in 6 well plate and 24 hour later transfer them to the 24 well plate (100K cells/ well) and collect cell lysate the next day.
In this way, I found around 80% confluency which is enough for RNA extraction.
Take care
Best wishes
Ayman
https://link.springer.com/article/10.1007/s00401-023-02555-3
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