8 replies to this topic

[Mati]

Hi guys we all know that HEK 293 cells are very easily detached on washing with PBS. I am facing the same problem, i culture cells with medium containing antibiotics.
For transfection i must use medium without antibiotics , so during PBS washing or even only medium change the cells get detached, how can i controled this situation. I cannot proceed my transfection.
Thanks

[Gangwolf]

Use ExGen (Pierce) or Turbofect in vitro transfection reagent (Fermentas) for the HEK293 cells. These transfection reagents can be added to medium containing both FBS and antibiotics. I use both of these reagents regularly in the HEK293 cells with very good transfection efficiency. Turbofect is slightly better but it is being replaced by ExGen and might not be readily available.
If you'd like a protocol, just let me know!

Edited by Gangwolf, 13 December 2011 - 11:53 PM.

[GNANA]

i do use HEK cells and i too hav to face the same prob when i change the medium...but still i do...if the confluency is over 75%(when they are in clumps), they tend to detach easily. so reduce the confluency on the day of transfection/medium change..and you got to be more quicker (change the medium immediately after you took the previous) and gentle (along the sides of the dish slowly) while changing the medium.  i follow these steps and i do lot of transfection in HEK...by the way i dnt give PBS wash so it is bit easier in my case.

[Mati]

@Enthusiast thanks for suggestion now i am using Fugene HD transfection reagent.If my transfection is not succfull i will use Turbofect.

@Veteran Thanks i will try now more gently.

[GNANA]

I am also using Fugene so you should able to end in a positive note....good luck.

[Rsm]

I am using good old Calcium phosphate transfection, works well (better than lipofection in my hands) and costs nothing. But I guess that's too oldfashioned...

Anyway, the best way to prevent detachment of HEK293 cells is to pre-treat the plates with a 0.01% solution of (mono) Lysine in PBS. Then you cells will not want to detach ever again, particularly if you use Trypsin/EDTA...

[Mati]

Thanks to all for your Suggestions
@veteran Usually how much transfection efficiency you got with Fugene HD, i mean to say did you transfect your cells very easily, in my case i already tried two times but no success, do you have some important key points for transfection and also i dont have any positive control so i can compare my samples with cotrol. Do you know any method that i can compare my transfection efficiency??

[Harvester]

You can easily check your transfection efficiency by transfectimg a GFP coding plasmid.
As for HEK293s detachment tendency, I try to avoid it by being extremely gentle when handling them and by washing them with warm PBS: I aliquot 50 ml of PBS in a falcon and let in a 37oC waterbath for 20 min.

Edited by Harvester, 15 December 2011 - 07:24 PM.

[Mati]

Thankyou Harvester i will try the same .