I transfected HeLa cells with 0.3uL of XtremeGene-9 and 100ng of GFP-tagged DNA. After two days I fixed the cells with 4% Formaldehyde in PBS for ten minutes, then washed with 0.1% Triton X-100 in 1xPBS, and blocked with 1xPBS, 1%BSA, 0.1% Triton x-100. The stains I used were Hoechst, and Concanavalin A for the ER.
I only see the debris in the Hoechst channel. I also don't see any nuclear debris on the same 96-well plate for cells which were not transfected.
Can anybody tell me what the debris is from and how I can get rid of it?
Hi,
Have you checked your culture for contamination? Maybe you have bacterial or mycoplasmen contamination? Were the nontransfected cells treated with the XtremeGene-9 in the same way like the transfected ones?
Dear Jessica,
Maybe one solution could be to try an alternative to your transfection reagent (less toxic and biodegradable) and check if the phenomenon still occurs.
Recently we have launched a transfection reagent dedicated to HeLa cells. HeLaFect presents a high compaction level of nucleic acid associated with low toxicity (biodegradable). More information are avaliable in the link below.
I would be very glad to send you a sample if you would like to try it; you can contact me via ResearchGate or directly at [email protected]
good luck with your experiment,
Cedric
http://www.ozbiosciences.com/transfection-cell-specific/77-hela-cells-transfection-helafect-reagent.html
The spots I was seeing turns out to be micelles formed by the transfection mixture (Transfection reagent + DNA + Opti-MEM).
I first tested my cells for mycoplasma infection using a detection kit to rule this out. My cells were not infected.
Then I added the transfection mixture to a well with no cells. I saw the artifact in this well, but did not see it in an empty well without the transfection mixture. What I am seeing are the micelles formed by the mixture.
I also tested just the transfection reagent alone added to cells, and there are no Hoechst artifacts. The same goes for just opti-MEM or just DNA added to a well. And any mixture of these (dna+opti-mem, dna+transfection reagent, opti-mem+transfection reagent...) do not cause any nuclear artifacts in the Hoechst channel. Only Opti-Mem + DNA + transfection reagent causes the artifact.
There are two ways I have used to remove the micelles. One way is to reduce the amount of transfection reagent and DNA (while still maintaining the 3:1 ratio). Another way is to change the medium after 6h up to 24h.
Figure: All wells on the 96-well plate were treated the same. The transfection mixtures were incubated for 15 minutes before I added them to the wells. I left the plates for two days before fixing with 4% PFA (in complete DMEM) for 10 minutes, washing with 0.1% Triton X-100 in 1xPBS, and blocking with 1xPBS, 1%BSA, 0.1% Triton x-100. I stained with DAPI for 1 h.
Nice images, Jessica!
I'm pretty surprised you saw these structures in the DNA + transfection reagent + Opti-MEM condition, but you don't see them with just the DNA and transfection reagents! I wonder what the media contains that is necessary for these micelle structures to form......
Do you add anything to the Opti-MEM media? Say, fetal bovine serum, glutamax, or antibiotics?
Thanks Jesse! I don't add anything to the Opti-MEM, but after I incubate the mixture I add it on top of cells in DMEM with 10% FBS. I use the protocol described by Roche.
Hi Jessica,
I think I have faced the same thing with transfecting HeLa ! I did transfection of RNA oligo and I found these debris on the wells that I transfected with high concentration of RNA but not with the ones of low concentration ! I'm still trying to find an answer ! and I'm wondering if these debris could affect the transfection efficiency ?!!!!!
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