I am using Optineurin gene and I want to establish stable cell line for specific gene expression. But when I have transfection using antibiotic resistance media, it almost died nearly 90%. SO after passage several time and change the media for remove the death cell. Most importantly, I used positive sample by Doxycycline. But it also showed not good efficacy in transfected cell. The gene was derived by using Flp-In system. When I used to transfection, the cell confluency was around 80% and I used following the method of Invitrogen Lipofectamine™ 3000 Transfection Reagent . Have any suggestion to get the good efficacy number of cell ?
Lipofectamine exerts toxicity. If you are using precisely the same volume mentioned in the manufacturer's protocol then indeed that is lipofectamine is the culprit which is killing your cells. You need to standardize (say serially dilute) to get the lower volume of lipofectamine followed by efficient transfection. it is highly suggested to avoid the use of antibiotics and serum.
Normally, lifofectamine 3000 should be 2x of your DNA/plasmid construct(DNA:lipofectamine = 1:2). Also, be sure to consider your cell number and DNA amount. The selection marker/antibiotic concentration should also be correct.
Hi Nazmul Islam
First, I would like to elaborate that transfection by Lipofectamine 3000 is not going to give stable cell line.
Second, Lipofectamine3000 is too toxic.
Third, to enhance the efficiency I will add some tips I hope they will give you a hand:
1. Adjust the ratio between DNA/Lipofectamine3000. Of note, 1:3 is the most useful one, but again it depends on your experiment itself.
2. Use a ratio of 1:2 DAN/P3000.
3. If you have too much toxicity, refresh the medium after 6 hours of transfection.
4. Try to observe the efficiency after not more than 48 hours because some plasmids are removed by the cells by the mentioned time.
5. For Lipofectamine3000 it is up to you to use medium with or without antibiotics and with or without FBS. You have to optimize your experiment related to those two factors.
All the best...
Mohamed Khashan ,
Actually I found some cell are colony formation. But when I added Doxycycline it showed only dead cell GFP. How can we confirm our transfection cell is correct without Dox ?
Salma Aktar normally I used to seed cell 1.5* 10^5 cells/mL in 6 well plate and after O.N growing started transfection. Sometimes I reduced the cell number 0.75*10^5 and put transfection with 48 h incubation. Then passage into antibiotics plate.
Hi Nazmul Islam
It depends on your next plan. If you would like to reuse the transfected cells for downstream procedures, then you can sort positive cells by FACS. If you are trying only to see the transfections efficiency you can use flowcytometry. However, in the mentioned procedures you have to tag your cells with GFP ... etc. Another possible way is to use RT-qPCR, so you can confirm a rise in the expression of your gene of interest.
Hope that was helpful.
Best ....
- Badges
- Science method