I am looking for a Lipofectamine 3000 protocol that someone has found to be very successful for HeLa. A protocol for Hek293 would be a bonus.
Here is my issue:
I did a number of parallel transfections with HeLa and Hek293 using SEAP reporters for a few transcription factors. I followed the protocol for 96-well plate transfections from the official protocol, including the part where washing the cells was not necessary.
I tested SEAP activity at 18-24 hours and found that my HeLa were expressing well, but my Hek293 were not yet producing in my positive control vector. I allowed my cells to incubate for 1 more night with the same medium (including the complexes, as I did not wash them away). At 2 days post-transfection my Hek293 are alive, but all of my HeLa transfectants have died. I believe this is due to the complexes, as there was no difference caused by choice of plasmid. All plasmids were endotoxin-free preparations.
If anyone has a tried-and-true protocol for HeLa it would be appreciated. I have already decided to wash them after overnight incubation, even though the protocol says it isn't necessary.
Suggest to request a new batch of Hela from other labs. HeLa should be quite easily to be transfected and resistant to the toxicity of transfection reagent. Sometime cell infected by microplasma can cause the failure and die after transfection. Although microplasma can be removed by drug treatment, but it takes time. Request a new one would be the simplest way to solve your problem.
Dear Jordan,
what is true for a cell type may not be true for another one meaning that, you are right a dedicated protocol should be working.
On an other hand it could be interesting to try an alternative to your transfection reagent: we have developed an HeLa specific transfection reagent, HeLaFect.
It comes with specific protocol ; you can have more information in the following link.
I would be very glad to send you sample if you want to try it ; you can contact me via ResearchGate or directly at : [email protected].
best regards.
http://www.ozbiosciences.com/transfection-cell-specific/77-hela-cells-transfection-helafect-reagent.html
HeLa are easy to transfect and typically require less Lipofectamine 2000 or 3000 - vs other "tougher" cells. so try using less reagent, and optionally incorporate media change at 12-24h
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