Hi, I’ve been using PolyFast transfection reagent from Medchemexpress (HY-K1014) and followed the general protocol. However I’ve noticed that my cells don’t look healthy afterward and I'm wondering what might be causing this. Could this be due to the reagent itself, the transfection conditions (e.g., DNA amount, incubation time), or perhaps something cell-line specific? Thank you in advance!
Hi Janice,
Indeed, cell viability and transfection efficiency strongly depend on a combination of the factors you mentioned:
Depending on the cell type and cell line, they have intrinsic ability (or not) to be transfected.
Then, also the transfection method and reagent used, the ratio of DNA: transfection reagent, the absolute amount of DNA will affect transfection efficiency and viability.
My advice is, if you have the possibility, to try different transfection methods and to optimize the parameters.
About DNA, not always more is best, as it also can be toxic on cells. Moreover, try to use a DNA extraction kit that gets rid of bacterial endotoxins.
Good luck and get in touch if you want to discuss further details.
Hy Janice,
If your cells are not in good condition after PolyFast transfection, several factors could be contributing to the problem. One of the most common issues is using too high a concentration of PolyFast, as it is a cationic polymer that can be toxic to cells when used in excess. It's important to follow the manufacturer’s recommended DNA-to-PolyFast ratio and, if necessary, reduce the amount of reagent or DNA used in the transfection. Another potential issue is the quality or quantity of the plasmid DNA. Low-quality DNA, especially if contaminated with endotoxins or salts, can severely affect cell viability. Always use endotoxin-free plasmid preparations and optimize the DNA amount to avoid cellular stress.
Additionally, the presence of serum or antibiotics during the transfection process can interfere with complex formation or increase cell stress. It’s generally advised to transfect in serum- and antibiotic-free medium and then replace it with complete growth medium after 4–6 hours. The health and confluency of the cells at the time of transfection also play a crucial role. Cells should be healthy, actively dividing, and at about 70–90% confluency. Over-confluent or stressed cells, such as those that are freshly thawed or have undergone many passages, may respond poorly to transfection.
Moreover, prolonged exposure to the PolyFast-DNA complex can be harmful. If the complexes are left on the cells for too long, consider shortening the incubation period and changing the medium afterward. Lastly, some cell lines are inherently more sensitive to cationic polymers like PolyFast. In such cases, optimization is key—this may include reducing reagent volumes, using alternative transfection reagents, or applying gentler methods tailored to your specific cell type.
Hope this may help uhh
Hello, Fan Janice
PolyFast (HY-K1014, MCE) has a certain degree of cytotoxicity, and it is normal for cell viability to decrease after transfection. It is recommended to use cells with a density of 70-90%, in the exponential growth phase, and a survival rate of more than 90% during transfection.
The answer to this question comes from MedChemExpress (MCE) Technical Support.
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