I am currently trying to transfect plasmids for expression of fusion proteins into ARPE-19 cells, and the Lipofectamine 2000 is causing a distinct morphological change. With or without DNA, the cells become filled with what appear to be vacuoles/large round bodies that are transparent (light/white) when viewed by transmitted light microscopy (see attached images) The number and size of the bodies appears to increase with the amount of lipofestamine used.
The bodies look like the lysosomal accumulation found in Yoon,2010 (see attached link, figure1). And indeed the cells appear sick. So I assume at this point this is some sort of cytotoxicity from the Lipofectamine 2000.
Does anyone out there have experience transfecting ARPE-19 cells with Lipofectamine or similar reagents? Have you seen this? How do you transfect your ARPE-19 cells to avoid this?
http://iovs.arvojournals.org/article.aspx?articleid=2127254
Hi! we have been using successfully lipofectamine LTX with plus reagent (also invitrogen/life technologies). The transfection efficiency is not great but we use it routinely with good protein expression, even to establish stable lines. for transient transfections i would suggest expression analysis 36h post-transfection, seems to be the best time in our hands, starting transfection at about 70-80% confluency. good luck!
Does the "bubbly" phenotype shown in the top figure above occur with LTX?
on occasion, we do observe some vacuolization of the cells, but never anything as pronounced as the pics you have here. But almost all cell lines (and i've worked with a few: HEK293, MBEC, HepG2, HeLa, ARPE-19, 3T3... just recently) display vacuoli-like structures during the first couple of days post-transfection with liposome-based reagents.
About ARPE-19, we've tried Lipofectamine, lipofectamine 2000, lipofectamine 3000 (invitrogen); Fugene6, FugeneHD (promega/roche); Attractene, Nanofect (qiagen). And the best results accounting expression/cell viability were with lipofectamine LTX with plus (1ug pDNA+200uL OPTI-MEM (or serum free medium)+2uL Plus reagent, incubate 5min RT, add 6uL LTX incubate 30min RT; drop-wise onto 1 well on a 6-well plate (about 3,5cm diameter); scale up or down as appropriate).
Hope that helps
Hi Floyd, one option would be to use a non lipid based transfection reagent. I can suggest Magnetofection which showed great efficiency in numerous cells including primary retinal ganglion cells and retinal pigment epithelium. You can find the info here:
http://www.ozbiosciences.com/content/13-magnetofection
Good luck
Olivier
Hi
Could you help me? I know that this is not the answer to Floyd. However I want to ask you something since you employ arpe-19.
I have noticed that growing these cells in Dmem/F12 + 10% SFB change their morphology and start to appear a round population like stem cells. Now O am trying to feed them only with dmem + 10% SFB... do you think that these cells can expand in this type of medium? What I can do?
Augustina,
I only worked with the ARPE-19 cells for a short time but we did grow them in DMEM/F12 +10%FBS and had the (to my knowledge) normal morphology as in the second picture on the original question. The only occasions where the majority of the cells rounded up and/or deteriorated were linked to dying cells. One occasion was a bacterial infection, and another had to do with a transfection attempt.
I second Eduardo's suggestion that a picture would be helpful to diagnose sick looking cells.
The accumulation of round intracellular bodies in the first picture of the original question ultimately was linked to transfection reagent (Lipofectamine) the more Lipofectamine I used the higher accumulation of the round intracellular bodies, which didn't necessarily lead to sick cells but these bodies did persist long term in the cells (greater than 2 weeks - I only kept the plate where I assessed the increase of intracellular bodies via dose of Lipofectamine for ~2 weeks).
Thank you Floyd but there not dying cells...they proliferate..accordiing to one paper...it could be a de-differentiation to stem cells and F12 would allow this......I do not know....
Eduardo makes a good point. Over the last ten years or so we in the cell biology world we have learned the hard way that we need to be very concerned about the identity of our cell lines, especially when they behave unusually.
It won't hurt to grow up some cells from another stock/lab/source to make sure they are behaving consistently with your current stock. You don't even have to stop growing your current stock to check this. Easy and provides peace of mind.
Have you ever perform Dapi staining? Do you see only the nuclei? I am thinking that their granulles have fluorescen at the same lamda than dapi. I check mycoplasm via PCR and rhey are free
If you are referring to the round bodies/granules from my original post, they ultimately were linked to aggressive lipofectamine transfection, so I don't suspect mycoplasma. However, I agree that mycoplasma can be an easily overlooked issue you want to remain vigilant about.
Now I have the cells growing corrctly with DMEM. I found a paper that indicate that F12 induce stem reound cell like ...
On the other hand...when I stained nuclei, in hRPE and ARPE19 cell lines I saw the like granules in the cytoplasm
Hi Agustina Alaimo . I have the same issue with ARPE-19 cell line. I did electroporation and selected the knocked-in cells successfully, but they undergo apoptosis. Do you mind please sending me the link of the paper, about the effect of F12 media on this cell line?
Uhh sorry I cannot find this paper 🙁..but taking into account my experience and talking with 2 colleagues in argentina, all of us conclude that it is so much better use DMEM (ONLY..NOT WITH F12) and add glutamax and 10% SFB. If you can use SFB Gibco at least to defreeze cell much better..but it is not the most important thing. I did not try to transfect them but I used to transfect cell with PEI. Good luck!
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