There is a major problem with transfections in our project. I have recently tested Lipofectamine 2000 and a Cyan Fluorescent Protein (CFP) plasmid on CHO adherent cells. According to the protocol, it should get us about 96% of cells expressing our protein. We have tried optimizing transfection, and ended up with pretty much the same parameters as recommended for said cell line.
Confocal images of transfected cells show that about 1% of the cells is transfected. My analysis points out several factors that may cause transfection failure. I will gladly accept any piece of advice from more knowledgable researchers on whether those factors can inhibit the process to such large extent:
1) I've been using DMEM without antibiotics that is possibly infected with bacteria. DAPI stained a lot of microbes on the slides.
2) The method I'm using is: seed the cells on coverglass like this: http://www.emsdiasum.com/microscopy/products/images/70000/70460-2R1.gif
next let them attach and proliferate in antibiotic-free medium for one day until they reach 80% confluence. To that I add the plasmid-Lipo (1:2) complex obtained according to the protocol using OptiMEM (100uL per well).
3) The viability of transfected cells is very low. They detatch and I loose more than half of them in transfected wells.
4) I fix them with ethanol buffered with PBS for 2 mins in -20 Celsius after 24 hours.
I don't know if any of the above can disturb lipofection. I've run out of ideas. These cells should be full of my plasmid, yet only a few are showing cytoplasm with CFP.
Hi, I think that most of the potential problems have already been well addressed, and if you have a contaminated medium you should absolutely fix that before considering any other problem. Anyway, when I transfect 293 cells, I do it in 12- or 6-well plates and to improve efficiency I plate the cells just before transfection and not 24h in advance. So during the first hours of transfection, cells are still "in suspension" and have more surface of contact with the Lipofectamine, which improves efficiency. I can forward you a protocol if you need.
Some basic questions: Have you been able to transfect other cells normally? Are you sure your CFP plasmid is functioning correctly? If you have an inverted scope, you could simply have the cells grow in a 24 well plate and visualize transfection without the need of coverslips - One less thing to worry about until you fix your transfection efficiency issue. Do you have to use CHO cells? 293 cells transfect really well.
Hey Jonathan,
I first tried with hard-to-transfect H9C2 rat cardiomyocytes. They failed and now I tried with CHO which are proven to be easily transfected.
I am pretty sure the plasmid works correctly, since I saw a couple of cells that were transfected and it worked before for other researchers.
I don't have to use CHO, I will transfect HEK 293 as soon as they reach confluence.
Yes the confocal has got inverted scope, but I'm not sure if the 24 well plate is compatible with it.
Dear Filip,
there are several points that need to be taken into consideration, and I'm sure we wil lfind something to enhance the transfection in your cells.
Let's go back to the points you've mentionned :
1) DMEM w/o antibiotics: the presence of contaminants in medium (for complexes formation or culture medium) can dramatically hamper both transfection efficiency and viability. It is really important to use a medium free of contaminants or at least, if you have no other choice, filter it on 0.22 µm.
2) conditions seem ok ; even if DNA quantity can be lowered to gain in viability (see below)
3) it's not so surprising since bacteria can damage the cells, and in presence of transfection reagent, cells become even more sensitive to contamination. Moreover, L2000 is known to be quite efficient but toxic (lot of DNA is also used).
The fact that cell detach indicates also that they are not in good shape.
4) this point should be ok.
I would highly recommend several options :
a. change your culture conditions: use an endotoxin and contaminant free medium for culture and complexes formation.
It is highly important that complexes are formed in medium w/o any supplement (serum, antibiotics, proteins, growth factor....).
b. the second option would be to use Magnetofection technology to enhance the efficiency of lipofectamine in these cells. Combimag (magnetic nanoparticles) reagent can be added to Lipofectamine to enhance its efficiency without changing the conditions. you can take a look to the work by Mukhtarov et al, 2008 and Wong J et al 2009, to confirm the role of magnetofection in CHO cells:
http://www.ncbi.nlm.nih.gov/pubmed/19439407
http://www.ncbi.nlm.nih.gov/pubmed/18632458
c. the third option would be to try an alternative to Lipofectamine such as DreamFect or DreamFect Gold: these transfection reagents are biodegradable and have high compaction level of nucleic acid and allow using less DNA: viability is enhanced:
http://www.ncbi.nlm.nih.gov/pubmed/20393662
http://pubs.rsc.org/en/Content/ArticleLanding/2011/SM/c1sm05838j#!divAbstract
If you want some supplemental info on the above reagents you can contact me at: [email protected], I will be more thant happy to help.
good luck for your experiments,
Cedric
One more thing. What promoter is controlling the expression of the CFP? Are you sure this promoter functions in CHO cells? We typically see expression in a handful of cells in a well when we use plasmids with promoters that do not typically function in the particular cell line. Following Invitrogen's instructions for transfection and the proper media conditions for 293 cells, you should be able to transfect 293 cells at 90-95%. Good luck!
Sometimes the transfection efficiency depends on the cell type also! Try doubling the dose for Lipo 2000. Also change media after 4-6 hrs post transfection with media without antibiotics. I agree with Dr Ploski that checking the promoter is important.
Cedric, thanks for your answer. Contamination is a probable explanation. I will try to get rid of bacteria. What you're saying is very true, I've also noticed that lowering the amount of plasmid has got drastic effects on cell vability. The complexes are formed in OptiMEM so I'm not worried about that. This Lipofectamine has worked previosuly for Luciferase assays in 96 well plates.
I have already tried optimizing Lipo and DNA concentration. After 6 hours I have tried to change medium for one with antibiotics and tried without changing it.
Jonathan, I believe it's CMV promoter. I tried a plasmid with SV40 promoter without any results as well. HEK transfection is on the way. Thanks.
Hi, I think that most of the potential problems have already been well addressed, and if you have a contaminated medium you should absolutely fix that before considering any other problem. Anyway, when I transfect 293 cells, I do it in 12- or 6-well plates and to improve efficiency I plate the cells just before transfection and not 24h in advance. So during the first hours of transfection, cells are still "in suspension" and have more surface of contact with the Lipofectamine, which improves efficiency. I can forward you a protocol if you need.
Andres, I will gladly read your protocol if that's possible.
The medium looks clean, but I will use a new one. The idea with having the cells in suspension is logical, but sometimes lots of the cells do not attach to the surface and therefore many transfected cells would be washed away. However, if that works with you, I will follow your protocol and test this out.
Hi Filip, here is my protocol for producong recombinant viruses in 293T cells. If you just want to express proteins, you have to follow the protocol until step 9, incubating for 48h instead of 24h. Don'to follow steps 10 to 12, and after 48h incubation you have to carefully discard the supernatant and resuspend the cells in a non-lysing buffer (15 mMMOPS,145 mMsodiumchloride, 2.7 mM potassium chloride and 4 mM calcium chloride, adjusted to pH 7.4) or simply observe them in the microscope.
Hello Filip!
I have one thing to add. I usually add an additional purification step by isopropanol precipitation for plasmids before cell transfetion. It helps to get rid of bacterial contamination. Maybe it will help you.
Good luck!
Andrés, your protocol works very well! And it's a very clever way to utilise the fact that Hek293 live happily both in suspension and adherent. I have incubated for 24h and got great results.
It's possible that having the cells in suspension and mixing them thoroughly with DNA-Lipo complexes enhanced the transfection.
Hi Filip, It's great to know your transfections work fine! Keep on going!
Dear Andres, I have the same problem in transfecting my cells. Can i borrow your protocol.
For sure Suresh.
I think I've uploaded a copy to the discussion thread, but if you can't find it don't hesitate to ask.
Hey Andres,
We are trying to express fusion proteins from CHO cells and we haven't been able to get much expression. Will your protocol be effective for protein expression from HEK293T cells too? Also we are using regular lipofectamine, do you think we should change our lipofectamine too?
Hi Deepak,
I'm not sure I've completely understood your question. My protocol was designed for (and tested in) 293T cells but if you want to try it in CHO cells I don't see any problem. As for the lipofectamine, I use Lipofectamine2000 so yours should be ok.
Good luck!
Hey Andre,
I had another question. Did you try to transfect your 293T cells by plating the cells 24 hours in advance? If you did, did you see a difference in protein expression levels or transfection efficiency?
Hi Deepak,
I used to plate them 24h in advance and it works quite fine in most of the cases. However, the "reverse transfection" in my protocole performed somehow better, mainly with a few "complicated" proteins.
Hi guys, I have been reading the comments above. I have been trying to express GFP in HeLa and HEK but have obtained no results on ECL film. I have used lipofectamine 2000. is there a specific L2000 to nucleic acid dilution ratio at which transfection can be enhanced? does anyone know any research papers where they have mentioned the failures doing anything similar? I have a submission in 8 days and need to know what all can go wrong. any suggestions please?
Dear Andres,
I have a few questions regarding your transfection protocol:
1. Is the 238 ul in step 1 per well or in total. Same question for the 16 ul Lipofectamine (I assume this is in total for 6 wells) and 60 ul of OptiMEM.
2. How much of the mixture do you add per well in step 8?
3. Approximately how many cells/mL is the 1.2 mL of cells you plate?
Thanks in advance!
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