Human dermal fibroblast cells are seeded to transfect using Lipofectamine2000 for generating iPSCs. plasmid/lipofectamine ratio 2:1. 15% FBS and pen/step antibiotic 500 microlitre per 100 ml.
Hello Chandra Kishore
Lipofectamine 2000 is toxic to cells. I would suggest you transfect the cells at 80% confluency. Expose the cells to Lipofectamine 2000 for a few hours only. Reducing Lipofectamine 2000 concentration also helps to reduce cell death. Usually DNA to Lipofectamine ratio of 1:1 or 1:2 works. Do not split the cells immediately after transfection. Let the cells recover overnight.
Best Wishes.
How long do you keep the mixture in the culture? 15% FBS is high while the cells are in transfection.
@Chandra Somasundaram. Mam, we keep mixture in culture for 4 hours.
Hi Chandra. It is hard to advice on this with limited information you provided. How is your actual amount of DNA and lipofectamine you used? 2:1 ratio is fine but also amount of each you use matters depending on cell culture plate size. Is that recommended amount for the size of plate and volume of cell media you used? Lipofectamine is toxic but shouldn’t kill cells for 4 hours of culture. Make sure you use relevant amount of DNA and lipofectamine for your plate size as too much lipofectamine may be toxic to cells if used at high amounts.
In our lab we use polybrene (polybrene conc. 4-8 ug/ml) which is a cationic polymer that can greatly enhance the efficiency of the retroviral or lentiviral infection to the mammalian cells. It acts to neutralize the charge repulsion between virions and the cell surface, thereby increasing infection efficiency
Masuma
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