Dear colleagues, should I change the medium after transfecting HepG2 cells with the RNAiMAX reagent for 24 hours? The supplier's protocol does not include this step in the knockdown assay, while some authors describe it in their methods in papers.
Generally speaking it is not necessary to change the medium if you're using chemical transfection techniques at low concentrations (see the protocol for HepG2: https://altogen.com/product/hepg2-transfection-reagent-hepatocellular-carcinoma/). There can sometimes be issues with the medium getting a bit messy, but usually the best way to approach such a situation is to add fresh, clean medium. After you assay for transfection results in a few days, then you can change the culture medium, and that should keep the cells healthy.
My fiance uses RNAiMAX a lot and she says that she did see it somewhere from Life Tech to change the medium 12-24 hours after transfection, but she can't remember where she saw it.
From personal experience, the lipids in lipofectamine tend to be really toxic to my cells and I always change the medium at the latest 1 day later. A customer service rep from Life Tech once told me to change the medium 4 hours after transfection to reduce the toxicity from the lipids.
Hi, Firstly I prefer to use reverse transfection method for HepG2 cell (i saw that it is more efficient). Secondly, i use media without FBS and antibiotic in the transfection moment.After 4-5 hours, i add more media with FBS (without antibiotic)(1 volume and 2X FBS,example if you use %10 FBS, add %20 FBS to make %10). then next day i change the media completely. I hope it will help you.
For transfection, use basal medium without serum or antibiotics; after 5-8 hours if possible, but definitely by 18-24 hours, add medium with 2X serum (without antibiotics)
Agree with Jordan. I use Interferin (not RNAiMAX), which is also a lipofilic transfection agent, and I change the medium the next day after transfection, because the clear toxicity is observed.
Dear colleagues, thank you all for sharing your experience. Helped a lot.
Whatever works! I think its best to try things out and see what works the best for you. There is no 'perfect' protocol and these things are dependant on various factors such as cell lines etc. In my hands, I have never had to change the media but I know others have had to. So I guess, just try it out. All the best.
I have used Lipofectamine (and I´ve changed media after 4-6 hours) and also Macsfectin (Miltenyi Biotec). In my opinion this one works better (and the protocol is even easier and shorter aswell) with higher percentages of transfection. I´ve replaced media 24 hours after transfection, aswell DMEM w/o FBS). Cells were HEK293. Good luck!
Hi Peter
I have used siRNA knockdown in primary cells (HUVEC) and cell lines as 293T. In my experience its better to replace transfection media with serum containing media after 3-4 h in case of primary cells. However, in case of cell lines, you can change media after 24 hrs in order to avoid cell toxicity.
Good luck!
I usually change the media after 4 hours of transfection and replace with complete media (10% FBS ). cells remain alive in case of HEK-293T
it's a common recommendation for all chemical transfectants in case of toxicity: change after 4h, and/or add supplemental medium after 24h. However, for many cell types, polymer-based reagents are less toxic than lipid-based reagents. Our Viromer BLUE for example gives very high KD for HepG2 without sign of toxicity. Bests, Sandra
Generally speaking it is not necessary to change the medium if you're using chemical transfection techniques at low concentrations (see the protocol for HepG2: https://altogen.com/product/hepg2-transfection-reagent-hepatocellular-carcinoma/). There can sometimes be issues with the medium getting a bit messy, but usually the best way to approach such a situation is to add fresh, clean medium. After you assay for transfection results in a few days, then you can change the culture medium, and that should keep the cells healthy.
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