9 replies to this topic

[labrat612]

I have perhaps asked this question before, but I'm totally stumped. I can't get my transient transfection to work in my suspension cells.
In order to just get the transfection parameters figured out, I've been using just a GFP plasmid.
Does anyone have a good protocol for this??

Please Help!!! I'm totally desperate  

[laurequillo]

labrat612, on 09 August 2010 - 09:32 AM, said:

I have perhaps asked this question before, but I'm totally stumped. I can't get my transient transfection to work in my suspension cells.
In order to just get the transfection parameters figured out, I've been using just a GFP plasmid.
Does anyone have a good protocol for this??

Please Help!!! I'm totally desperate  

I used to work with Jurkat and with Lipofectamine. One trick was to do the mixing of the Dna and the Reagent in a glass tube, not in a plastic one, and then add the mixture to the cells. I will check to see if I did something else...

[laurequillo]

Here the protocol1 For Jurkat Cells:
- Wash the cells 2x PBS
- Seed 10⁶ cells in 300 ul warm Optimem in 24 wells/plates.
- in a luminometer tube mix 2.5ul lipofectamine and 22.5ul Optimem (per transfection point)
- In another tube mix 1ug of plasmid and up to 25ul with Optimem
- Add the lipofectamine mixture to the DNA tube, slowly and pippeting up and down.
- Incubate 30 min at RT
- Add 150ul Optimem to every tube (150ul per point)
- Add the 200ul to the cells (200ul per well)
- Incubate 6-8 hours at 37ºC.
- Add 500ul complete RPMI (per well) and let them rest for another 16-48 h.
- Centrifuge cells, remove supernatant, and seed them in RPMI.

I hope it is clear (It´s been a while since I did it, so I cannot be more specific!)

[labrat612]

thank you soo much for sending this.

Just to clarify: at the last step, when you replace the media with RPMI, you keep the cells in the 24-well plate, correct?

[laurequillo]

labrat612, on 13 August 2010 - 10:50 AM, said:

thank you soo much for sending this.

Just to clarify: at the last step, when you replace the media with RPMI, you keep the cells in the 24-well plate, correct?

The point is that I dont really remember... , but I think once you have them transfected you can seed them in a bigger well if you want (or maybe combine 2 wells with the same transfection...)

[genehunter]

If you still have no luck, you are better off to use electroporation. Most lipid reagents dont work very well with suspension cell lines.

[k8e]

Hey Labrat,

I have also had problems transfection suspension cell lines, they can be quite tricky.. I found that by spinning my plates downs once I'd added the transfection reagent I got slightly higher transfection efficiency, so this may be worth a try..

If you have access to an electroporator I would highly recommend going down that path.. It will save you a lot of time and although high cell death, it tends to work very effectively for most suspension cells.. It might save you a world of pain!

What cell line are you trying to transfect?? I had problems with mine and eventually it found out they were resistant to chemical transfection.. Painful!

Cheers and good luck!

Katie

[labrat612]

Hey Katie,

I'm trying a few cell lines to determine which would work best, at least in my hands. I'm currently working on CHO-S cells to see if they can't express well. I haven't come across any literature to say that they are resistant to chemical transfection.

I'll try your idea of spinning down plates following the addition of your transfetion mix. It can't hurt!

Thanks!

Edited by labrat612, 03 September 2010 - 06:15 AM.

[briguy7]

If you have the money, try Invitrogen's freestyle max transfection reagent--it is really expensive but I have found it works really well for suspension CHO-S cells (and for DG44 CHO cells)

[KevinK]

We have reports of FuGENE HD working well in CHO-S cells in traditional as well as defined media.  You can find the protocol in our online database here: http://www.promega.c...s/FugeneHdTool/

Feel free to request a free sample to see if you have the same success: http://www.promega.c...ection-reagent/

Kind Regards,
Kevin