I will be using COS7 cells to study some miRNA and gene interactions using Luciferase Assay.
I need advice from people working with these cells. When I start a culture from stock, can we use the cells for transfection after growing them overnight? I have worked with Sy5Y cells before and we have to wait until the 3rd or 4th passage to do anything with the Sy5Y. But COS7 I believe can be used immediately after overnight culture, right?
Any other tips on working with these cells are also welcome. Thanks!
I think generally you should pass cells a few times before transfecting. When cells are thawed they grow very slowly and are more sensible than cells which have been passed a few times. Transfection is a harsh process and you must assure your cells are at their best when you're going to perform such technique.
I agree with Ana. You should culture and expand cells for at lease 1 week, before you do transfection.
Dear Shruti,
As others, we generally do not recommend transfecting cells the day after they were thawed: this may results in elevated toxicity and lowered efficiency.
Moreover it is generally recommend to transfect any kind of cells when they are in their linear exponential growing phase. And for that, they need to be passaged at least 2 or 3 times and seeded 24H before transfection at a confluence so that they will be 70-80 % visual confluence at the day of transfection.
You can use DMEM medium - 10 % SVF - LGlu, P/S, for culturing and also for transfecting.
Concerning the transfection part, I would highly recommend a new reagent dedicated to COS cells: COSFect. It is a transfection reagent based on biodegradable properties that present a high level of transfection. For more information you can have a look at : http://www.ozbiosciences.com/transfection-cell-specific/76-cos-cells-transfection-cosfect-reagent.html.
I would be glad to send you some sample to test, and if you have any question please do not hesitate to contact me at : [email protected]
good luck with your experiment,
best regards,
Cedric
Hi,
As per previous comments, transfecting new cells in layman terms is toxic. Cells will die and you will not reach the expected transfection efficiency. At least one or two passages make them acquainted and they grow well. At P3 or so, u can seed at a good density so that by next day they are confluent enough for you to transfect. Transfection efficiency can vary from cell line to cell line but as far as I am aware, COS can be transfected at 70%.
Kalpita
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