I need a quite a large number of cells to check the expression of certain constructs. Right now I do not have that many cells growing. Can I thaw a frozen vial and without passaging, use them for transfection just to check the expression? I know freshly thawed cells should be passaged at least twice before they are used for any experiment, but in this emergency, can this be done?
In theory: maybe it will work. Depending on how you transfect, they might die in the process.
Also, as you said, one shouldn't use freshly thawed cells for experiments..you will not know how it will effect your experiment.
Dear Tias,
in theory, it can be done. practically, results will not be reliable.
The point is, cells need to be in their exponential growing phase to be considered as "in their natural and best shape". You surely have noticed that if you wait too long, cells will be squeezed by other cells, stop growing and arrest every metabolism process. (cells that will not stop will detach and go under apoptosis).
In the contrary, if cells have not engaged their metabolism, they will not present their full potential. And thus results will not be significative. Transfection should occur, but mortality should be high and maybe efficiency, low.
This is why we always recommend passaging cells at least twice and use them in their exponential growing phase before transfecting them.
I would take the opportunity to present you our transfection reagents that are really efficient, and biodegradable. Their high compaction level of nucleic acids allows using less material for a better viability.
If you want to try some sample, please do not hesitate to contact me via Researchgate or directly at [email protected]
Cedric
http://www.ozbiosciences.com/
I wont suggest that, thawed cells under lot of stress and they tenu to have hard time to adapt to the media. It can be done but the results would be disappointing. You have got to give them atleast 2 subcultures before you proceed. If you are not planning on to publish the results nad just wish to check for expression of your construct, you can give it a shot
All the best
Dear Tias,
You have to wait at least 2 passages before transfection for significant and reliable result. But your thawed cells are not growing fast, right? Here is what you can do:
1. People generally thaw cells in T25 flask, 60mm/90mm dishes. But in your problem, thaw them in 35mm dish or better use a well of 12well/ 24well plate. This way you will see that your cells will be growing in full potential.
2. After the cells reach near confluency, transfer all the cells to bigger dish (24 well to 35mm or 35mm to 90mm dishes)
3. Seed exponentially growing cells from these bigger dishes to your desired size of plate for transfection.
Dear Supratim,
I know I should passage at least twice before I use for any experiment. I already mentioned that in the question, and the problem isn't of the cells growing slow. I generally plate them in 6 well plate and let them become confluent until I shift them to 100mm dishes. Thanks!
according to my experience, i found the cells (Stem cells) best for transfection when they are 60% confluent.
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