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!

 Isn't this reverse transfection?

Yes, it is.

Hi Filip,

Do you change the culture medium 6 to 12h after adding the DNA-lipo complexes? Also Lipo works better in medium without serum.

we transfect CHO here in the lab, and although we use Turbofect, we get very nice transfection rates even with very large cDNA. We change the culture medium immediately before adding the DNA precipitates (this could help the complexes crossing the cell membrane), and then after 6 to 12h we change the medium again (this improves cell viability, and help you keep transfected cells that are more fragile).  Although, in CHO, it worked well even without changing it after, but this for turbofect.

 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.

Hi Andrés,

Can I ask for your protocol as well? It will be a great help. Thank you.

Here it is Jinyue pu.

Good luck!.

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?

I use lipofectamine for more difficult to transfect cells (for HeLa I use Turbofect, and it works just fine). But when  I use Lipofectamine I use a ratio of 2ug of cDNA to 8 uL of Lipofectamine. I dilute 8uL of Lipo in 100 uL of medium without serum or antibiotics and the 2ug of GFP in 100 uL of the medium without serum or antibiotics. After 5 minutes, I mix the diluted lipo and the diluted GFP, mix gently, and incubate for 20-25 minutes, RT. It helps if you change the medium of your cell culture for medium without antibiotics, in this case the described amounts are indicated for six-well plates. So I replace the culture medium with 800 uL of medium with serum but without antibiotics, and add the lipo:DNA complexes to the culture. It also improves cell viability if you replace the cell culture medium again 6-12 hours after, and in this case you can use the medium with antibiotics again.

You should also check the quality of your cDNA!

hope it helps

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!

Hi Kylie,

Questions 1  and 2 : The 238 optiMEM + 16 LF + 60 optiMEM w/plasmids is the volume per well, so you add everything in one well. You can test one different palsmid combinations and/or replicate per well. 

Question 3 : For one 6-well plate, I start from one F75 flask at 80-100% confluence, which usually represents 8-10E6 cells in total and hence 1-1.2E6 cells per ml after resuspension in 8 ml.

Hope this info is of help.

Regards,

Andrés.