hep-2 & Hep-G2 cell line - difference between hep-2 & Hep-G2 cell line (Dec/15/2004 )

Pages: Previous 1 2 3

QUOTE (kalyani @ Jan 5 2005, 06:38 PM)

I have had problems growing HepG2 cell line. They do not grow very well as a monolayer, instead they tend to clump up. I use only T-25 plates and not any coated plates. Can anybody tell me how to avoid this problem?

I have had similar problems with MCF7, at low passage numbers (from ATCC), but as the passage number increases, they grow as monolayer than as clumps.

Any help is appreciated.
Thanks.

I only recently started to work in a Hepatology department but HepG2 cell culture and transfection is fairly standard here.

So far I've had no problems whatsoever growing HepG2s in DMEM, 10%FBS, Pen/Strep, L-Glu. Apparently the HepG2s can be acclimated to certain mediums, ie if you suddenly change the medium you will have problems, so maybe anyone with problems should check what the cells were grown in before they got them?

The way we get around the clumping problem is to squeeze the cells through a 25 gauge needle for 3-5 times after trypsination and before plating (if you need to count the cells, do it both before and after counting).

According to the experienced people working here, Lipofectamine2000 is the best transfection agent. Cells need to be seeded at 1x10to6 cells per well in 6 well plates and transfected when ~90% confluent (24-48h after seeding). Around 1-2ug plasmid and 10ul Lipofectamine and you can leave it on overnight. Using Opti-MEM for mixing up the complexes is supposed to be best.

Haven't tried transfecting my own HepG2s yet, but hopefully this info should help you.

Thanks to whoever mentioned the reverse transfection thing, I'll definitly give that a try!

-LeserattePD-

I have now transfected my HepG2s using the reverse transfection method. I've done some optimisation and had best results (>80% I'm not kidding!) when using 3ug oligonucleotide (oligo is Cy5 labelled) and a 1:2 ratio, leaving the transfection mixture 3h to incubate.

If anyone wants to see the raw data please email me.

LeserattePD

QUOTE (Hooly @ Feb 18 2005, 06:22 PM)

I just read about a technique called 'reverse transfection' for getting siRNA into those difficult-to-transfect cell lines. Basically this means that instead of plating the cells and leaving them alone for 24 hours before transfecting, you resuspend them after trypsinisation in siRNA/liposome soup and *then* plate them.

Haven't tried it, but it was in a newsletter from Ambion - you can find the details at the following link:

http://www.ambion.com/techlib/tn/121/9.html

They specifically say that this is a great way to get really good delivery/silencing in HepG2.

-LeserattePD-

HI,

I just start to work on HepG2 cell transfection with DNA. The reverse transfection you mentioned here is very interesting. Could you please provide more information, such as the transfection reagent, the incubation time,...? Thank you very much for your time and help.

Xin

QUOTE (LeserattePD @ Jun 6 2005, 10:27 AM)

QUOTE (Hooly @ Feb 18 2005, 06:22 PM)

-capricy-

Pages: Previous 1 2 3