Hello everyone,
Hello,
I tried recently to transfect the T24 cell line using the Lipofectamine 3000 and the Amaxa nucleofection. Unfortunetly, 24 hours after the transfection 90% of cells were apoptotic which means a high cytotoxicity despite following protocols offered by companies.
Is there anyone who has been successful in transfecting the T24 cell line?
Best regards.
Hamadi.
Dear Hamadi,
As far as I understood Amaxa Nucleofector is the electroporator for transfecting cells with a settled impulse charge. This is being performed without transfection agents in glucose/sucrose-enriched for cell membranes protection serum-free media. Transfection agents are designed as positively-charged lipids and other polymers with the target to bind and deliver negatively-charged plasmid DNA/RNA inside vital cells. In general the better is transfection agent, the higher is its positive charge and cell toxicity. Therefore lipofectamine, for example, is being used in very low concentrations for a small amount of DNA effective delivery. As DNA itself is non-toxic, decades-folded plasmid concentrations are often used for electroporation.
These two transfection approaches cannot bring benefits in combined application. So, if you used them together cell toxicity result is a very likely outcome. Then try to make both protocols or just one of them separately, and it will be OK independently on which cell culture you use for your transfection.
Best regards,
E.F.
Dear Hamadi,
As far as I understood Amaxa Nucleofector is the electroporator for transfecting cells with a settled impulse charge. This is being performed without transfection agents in glucose/sucrose-enriched for cell membranes protection serum-free media. Transfection agents are designed as positively-charged lipids and other polymers with the target to bind and deliver negatively-charged plasmid DNA/RNA inside vital cells. In general the better is transfection agent, the higher is its positive charge and cell toxicity. Therefore lipofectamine, for example, is being used in very low concentrations for a small amount of DNA effective delivery. As DNA itself is non-toxic, decades-folded plasmid concentrations are often used for electroporation.
These two transfection approaches cannot bring benefits in combined application. So, if you used them together cell toxicity result is a very likely outcome. Then try to make both protocols or just one of them separately, and it will be OK independently on which cell culture you use for your transfection.
Best regards,
E.F.
As mentioned above, the combination makes not much sense. T24 cells can be easily transfected using the classical calcium precipitation method.
It occurred that Hamadi made two methods independently from each other with the same cell death toll. For me calcium precipitation method is acceptable for lenti- and retroviral transductions. For classical plasmids I prefer polyethylenimine or lipofection which is faster and provides higher non-genomic transfection rate.
Mr Kay-Dietrich Wagner,
Could you provide a successful protocol for T24 transfection using the calcium phosphate method?
Dear Hamadi,
One of my original country mates Dr. Alexeyev posted a detailed CaPO4 transfection method description into one of our previous discussion:
thttps://www.researchgate.net/post/Is_Lipofectamine_3000_more_efficient_in_generating_Lenti-viruses_in_293FT_cells_than_the_CaPO4_methods
But if you are determined to develop into a skillful and equipped with laboratory practice postdoctoral researcher it is worth to learn to handle different methods, for transfection/transduction too. First, it is good to be able to choose a suitable method for every current task you would have with different vectors and cell cultures. Second, 95-98 percent of non-working in 2nd phase medicines number might be suppressed a bit if more guts are able to find mistakes and wrong-positive results in their own and the others research.
Cheers,
E.F.
Thank you Elena for your kind comments. Actually, the amaxa recommended program was very harsh to cells(T-030).So, I have changed it to a softer one. Currently, the transfection efficiency is very low but it will be better in few hours.
Best regards,
Hamadi
I have one self-developed hint for electroporation. I never use equipment with pre-installed software which is not programmable or possible for manual operations because I make lower than maximum voltage but doubled charge manually within 1-2 seconds. Cell membranes are often getting more permeable for the second discharge this way. And of course you should protect culture in dielectric medium, sucrose 10 percent solution is recommended for it. I make hybridomas fusion and direct retroviral transduction (without packaging cells) with it.
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