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suspension cell transient transfection


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#1 labrat612

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Posted 09 August 2010 - 09:32 AM

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 :unsure: :(

#2 laurequillo

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Posted 09 August 2010 - 09:54 AM

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 :unsure: :(


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...
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#3 laurequillo

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Posted 13 August 2010 - 05:29 AM

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 37C.
- 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 (Its been a while since I did it, so I cannot be more specific!)
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#4 labrat612

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Posted 13 August 2010 - 10:50 AM

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?

#5 laurequillo

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Posted 13 August 2010 - 09:33 PM

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... :unsure:, 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...)
"He must be very ignorant for he answers every question he is asked" Voltaire

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#6 genehunter

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Posted 14 August 2010 - 07:34 AM

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

#7 k8e

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Posted 01 September 2010 - 11:53 PM

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

#8 labrat612

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Posted 03 September 2010 - 06:13 AM

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.


#9 briguy7

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Posted 20 September 2011 - 09:28 AM

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)

#10 KevinK

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Posted 21 September 2011 - 07:17 AM

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
Promega Corporation
Madison, WI

#11 et2b

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Posted 26 August 2013 - 12:26 AM

Hi all,

 

I am working with Fugene HD to transfect Jurkat, but having no success. 

We tried vector/reagent mixtures from 1.5:1 till 8:1 and 3x105 and 5x105 cells, and incubation of 16 and 32 h...

nothing is working.

 

Any suggestions?

 

best






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