Mysterious case with a tranfection - could anybody give me a clue? (Sep/08/2007 )
This morning I transfected my COS-7 cells as usual, with 15 ul of lipofectamine diluted in DMEM and 6 ug of DNA for 60 mm plates (1/4 of the recommended amount by the protocol, but it used to work very well). The cells were at confluence and ok.
After six hours, when I was going to replace the medium for a fresh new medium with antibiotics, all the cells were detached, simply as if they were been trypsinized!!!
The lipofectamine was new, as I have started a brand new tube. The plates also shared the serum, medium, incubator and PBS, but the effect on the cells (the detachment) didn't seem a conventional contamination...
What do you think it could have been? A rare form of contamination, or what? 
After six hours, when I was going to replace the medium for a fresh new medium with antibiotics, all the cells were detached, simply as if they were been trypsinized!!!
The lipofectamine was new, as I have started a brand new tube. The plates also shared the serum, medium, incubator and PBS, but the effect on the cells (the detachment) didn't seem a conventional contamination...
What do you think it could have been? A rare form of contamination, or what?
May be ur cells were previously under some sort of stress(pH shock etc), not apparently visibile and upon transfection using lipofectamine, which is usually lipid based and results in some cell death; cells already being in a bad condition, faced subsequent shock and resulted in cell death and their detachment.
R u sure that media which u used wasn't too much chilled and was at ~ 37*C. well i'm not sure , bt may be this may be the reason
If there is bacterial contamination u will see some turbidity.
Any possibility that you used Ca2+, Mg2+ free buffer instead?
By the way, you probably want to use Opti-MEM or RPMI-1640, rather than DMEM as the medium during transfection.
maybe you could plate 0.5% gelatin on dish, we also observed this phenomena, after we plated gelatin, the cell attached to dish well.
After six hours, when I was going to replace the medium for a fresh new medium with antibiotics, all the cells were detached, simply as if they were been trypsinized!!!
The lipofectamine was new, as I have started a brand new tube. The plates also shared the serum, medium, incubator and PBS, but the effect on the cells (the detachment) didn't seem a conventional contamination...
What do you think it could have been? A rare form of contamination, or what?
could have been bacteria the problem?
Thank you for the answers.
It's not a problem of gelatin on the plates, as these cells are perfectly attached in all conditions...
I used to work with OPTIMEM but I've also work with DMEM and transfections are ok.
My colleagues and I suppose it could be a yeast contamination of the PBS...
So remember...It's wise to keep an individual aliquot of the PBS at 4ºC to avoid fungi or yeast growth!! 
You must have some super yeasts around you or your buffer were loaded with them. I dont see how could you get enough of them within 6 hrs such that got your cells killed.
I once had a similar problem, found out that the DMEM i used didnt have glutamine. Check if the DMEM is fine or anything out of ordinary. Also sometimes higher passaged cell lines could be the problem.
Did you by any chance do the transfection with DMEM containing antibiotics?
After six hours, when I was going to replace the medium for a fresh new medium with antibiotics, all the cells were detached, simply as if they were been trypsinized!!!
The lipofectamine was new, as I have started a brand new tube. The plates also shared the serum, medium, incubator and PBS, but the effect on the cells (the detachment) didn't seem a conventional contamination...
What do you think it could have been? A rare form of contamination, or what?
By the way, you probably want to use Opti-MEM or RPMI-1640, rather than DMEM as the medium during transfection.
hi
can u give ur protocol for using gelatin?
tnx