Extensive Cell Death with Serum Free Media - How to Avoid Extensive Cell Death with Serum Free (Aug/31/2005 )

I have been doing transfections that need to use serum free media. The problem is: 1~4 hours serum free media treatment itself is associated with extensive cell death . Lage scale (>50%) adherant cells detach from the plate and float to the top of the media. This happens to all three kinds of cells I use, namely HepG2, HEK293T and HeLa cells. The media I used so far are MEM, DMEM or Opti-MEM. For sure that this is not related to any contamination because all cells grow extremely well in full media. Even after the extensive cell death, the remaining cells can grow normally, only take more time. I was wondering if any one knows what is the problem? and how to avoid it? Thank you very much for your help!

Rosi

Hi

I do transfections using Fugene, that suggest that serum free medium is important, hoevere if you look at the data sheets and protocols that come with the reagent, then their data show about a 1-5% decrease in transfection efficiency in serum containing medium.

perhaps you could try transfecting in serum containing medium and see what happens.

BTW, we dilute our fugene and DNA in PBS rather than SFM.

Bob

Hi Rosi,
I transfect my NIH3T3 cells using Fugene 6 (Roche) and as bob mentioned above, I don't change to serum free medium, and the decrease in efficiency is minor and not significant. Also, when I use HEK293 cells I transfect them with Calcium phosphate (which we make ourselves in the lab) and in this method you 1) do not need to change to serum free medium, and 2) you need to wash the cells with PBS to remove the CaPO4. In 293 I get very high transfection efficiency, plus it is much more cheeper than Fugene

have you put 10 to 100 fold less antibiotics in your serum-free medium?

Seb_

We have been successfully using Gencarrier-1 for 293, HeLa and HepG2 trasfection in the presence of 10% serum (getting lazy to change to serum-free medium). The efficiency is not compromized nor the cell viability. You may want to try it.

Dear Rosi,

I think your problem might not be serum-free medium, but antibiotics in that serumfree medium. Depending on which transfection agent you use, that can kill your cells very easily.

I generally use serum-containing but antibiotic free medium plus Opti-MEM to dilute the transfection complex works just fine for my HepG2s. I use Lipofectamine2000 to transfect oligos with good success by something called reverse transfection.

Hope that helps

LeserattePD

I have been doing transfections that need to use serum free media. The problem is: 1~4 hours serum free media treatment itself is associated with extensive cell death . Lage scale (>50%) adherant cells detach from the plate and float to the top of the media. This happens to all three kinds of cells I use, namely HepG2, HEK293T and HeLa cells. The media I used so far are MEM, DMEM or Opti-MEM. For sure that this is not related to any contamination because all cells grow extremely well in full media. Even after the extensive cell death, the remaining cells can grow normally, only take more time. I was wondering if any one knows what is the problem? and how to avoid it? Thank you very much for your help!

Rosi

hi
first in transfection assay is to avoid any antibiotic during the transfection procedure. This decrease efficienc and kills many cells. Second one, it's advised not to use serum in medium, but in protocols, they say you can use it if your cells don't support serum free medium.
Alternatively, you can use 0.2%serum medium. Serum concentration is enough too decrease significantely the serum-starvation-induced cells death.

But keep in mind that death of cells is a marker of good transfection efficiency too...

fred

Hi Nekko,
The protocol I use is the one from a recipe book we have in the lab ("the red book") called Current Protocols in Molecular Biology. It's in Unit 9.1 of supplement 36. essentialy we replace medium 2-3hrs prior to transfection, to regular DMEM - INCLUDING antibiotics and serum as usual. Then, for 6wells plate (35mm) I prepare 90uL sterile DDW minus DNA volume; 2ug DNA (better to use 1ug/uL conc. if possible), so now the final volume is 90uL. To this I add 10uL of 2.5M CaCl2 and mix by pippeting. Now, in another tube (I prefer a 10mL conical tube) I add 100uL of 2X HeBS (Hepes buffered saline - see below). Now I add the DNA/Ca solution dropwise, and after 3-4 drops I vortex for ~1sec. I let it stand for 20 min. in RT then I add the mixure to the cells dropwise. about 6hrs later I wash the cells twice with PBS and this is time 0. 24-48 hrs post-transfection in perform my assay.

X2 HeBS concists of 0.28M NaCl; 0.05M HEPES; 1.5mM Na2HPO4. Titrate to pH 7.05-7.12. Usually it works for me at pH 7.05, but you have to check each new batch. An exact pH is extremely important for efficient transfection.

Hope I helped

PS: This protocol works best for 293 cells. for other cells you may need to extend the incubation time before you wash the cells.