PBS causes cell death in transfection - (Nov/23/2005 )
hello
I have some basic doubts.During transfection about 70-80% cell growth, when I add PBS(phosphate buffered saline,0.1 M,pH 7.4) to the cells and leave for 5 hrs.I found a whole big empty place in the middle of the 24 well plate.Later I replace the PBS with 2X medium.I find the cells growing back normally after 24 hrs.What causes the cell death during the first 1- 5 hrs?how could it be explained?
hi
correct me but : in transfection you should use medium wothout serum and antibiotics for the mentionned 1-5h?...
PBS doesn't contain any nutrients. So, you starve yourselfs. Also, Fred is right, most transfection protocols advise the use of medium during transfection.
Electroporation can be performed in PBS, but that's because you put your cells in pbs, add your DNA, electroporate and add back to medium in 45 minutes at most.
hi yep transfection needs to be done in medium free of antibiotics.But mine is the collected samples from polymer films which will have the PEI/DNA in it.so it should resemble the physiological solution invivo.So i use phosphate buffered saline.Probably i should try using phosphate buffered solution w/o NaCl.It should prevent osmotic shock in cells,as saline is 0.9 % Nacl,about 150mM Nacl, which causes osmotic shock.
hi
i think that 9%NaCl is good. It corresponds physiologically to 0.307 osmoles per liter, so it's very close to 150mM of NaCl.
Hence 'm not too sure you enounter a osmotic shock...
i think that 9%NaCl is good. It corresponds physiologically to 0.307 osmoles per liter, so it's very close to 150mM of NaCl.
Hence 'm not too sure you enounter a osmotic shock...
yep it looks loke phosphate buffered slution is an isotonic solution.so it shouldn't create an osmotic shock in animal cells.So probably I should search for other reasons
Are you adding a very small amount of PBS because you want to save transfection reagent?
If cells in the middle of each well are dying but cells near the edge are fine, it could be that the PBS is drying up over the 5 hours. Cells near the edge are protected from drying up by surface tension (the meniscus).
How adherent are your cells? my 293T cells are dislodged when medium is dripped onto them.
i agree with debbiek - if your cells are dying right in the middle of your well, maybe consider how you are applying the solution to the well