I am trying to transfect the gene of interest into HeLa cells using Lipofetamine 2000 to see if this gene would cause any changes in senescence ratio (using Senescence Associated beta-gal staining) and cell cycle profiles.
In order to maximize transfection efficiency, I have determined the plasmid to lipofectamine ratio should be 0.6:3 OR 1.2:3.6, according to the western-blot result. However, the cell toxicity induced by lipofectamine is a big issue in senescence measurement.
Is there any suggestions that you can offer to help reduce the cell toxicity while maintaining the gene transfection efficiency? How about transfecting the gene twice, at half amount of requested amount each time?
You can change the cell medium after 5hrs post-transfection to reduce the cell toxicity. Also Lipofectamine 2000 is very easy to use, it doesn't require certain DNA/reagent ratio. The volume of reagent you use is based on the plate type. For example, if you transfect Hela cells in 96-well plate, you need use 0.2ul of lipofectamine 2000 for each well, the plasmid amount can be various but don't pass the maximum --- 200ng/well. For other plate types, the amount of reagent you need can scale up from 96-well.
Thanks for your answer. Actually I did change the cell medium to DMEM, 10% FBS and no antibiotics.
Wang, the transfection was performed in a 6-well plate. I have already determined that by transfecting 1.2ug per well, together with 3.6ul lipo 2000 at about 50% cell density leads to the satisfactory target gene expression level while cells remain relatively intact. Although 1.2ug/6ul could achieve even more expression level of my target gene, the cells were in poor condition by 48hrs post-transfection.
So my concern was whether tranfecting 0.6ug plasmid for twice could leads to better cell condition with little sacrifice on the overexpression level of my target genes? Anyone tried this? Please provide your experiences..
Su,
you're using HeLa and Lipofectamine 2000, this combination has been used many times and it worked well almost every time. In other words, look for other issues such as:
- your lipid (lipofectamine) concentration
- your DNA/lipofectamine incubation period (should be done in serum free media such as OptiMem) - mix and incubate for 20 minutes
- your lipoplexes/hela incubation time (serum free media such as OptiMem until you take off the transfection media and replace it with complete DMEM with no antibiotics) 4-5 hours
what negative control are you using and how did it go?
Dear Bokhari,
Thanks for your answer.
The expression level of my target genes were really satisfactory.
Somebody in my lab suggest me transfect with lower cell density and less transfection reagent, in order to reduce cytotoxicity.
The negative control was lipofectamine+empty vector with the same backbone. Also cause significant cell death. The cell death event was evidently positively correlated with the plasmid that was transfected.
I have tried consective transfection: 0.6ug at day1 plus 0.6ug at day2, and harvest them/SA-beta-gal staining at day 3, to see if this provides any better results than single transfection of 1.2 ug. I will report to you guys when I got the results.
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