Trypsin:EDTA cell detachment process - (Apr/24/2006 )

Hi,

I used trypsin/EDTA to detach cells from their monolayer, but am unsure of the action of the trypsin and the EDTA in this process. Could anyone shed some light on the subject for me?

also my tutor doesnt use anti fungal drugs in his growth medium. Any ideas why he would do this?

Many thanks

dave

ps, the cell line used is U87. cheers

as good as I know:
EDTA - bind calcium (or magnesium?) ions that inhibit trypsin
trypsin - hydrolyse intracellular junctions

Most people don't use anti-fungals in thier media unless they have a problem, or if you also work with cervisiae in your lab and have had problems with frequent contamination.

Yup-

EDTA mops up divalent cations

Trypsin cleaves peptide bonds at lysine and arginine residues, except if the bond is to proline (?)

A lot of people don't use antifungals or antibiotics in their standard cultures, as the presence of those can mask low-grade infections.

to break cell-cell adhesion, you have to interfere with cadherin-mediated cell-cell adhesion. In the presence of calcium, these molecules are resistant to trypsin. If you remove calcium, by chelating it with EDTA, the cadherins become susceptible to trypsin, and you can get single cells.

I agree that anti-fungals are best omitted if possible, you never know the effects they may have on your cells and if you work in a clean way, they are not necessary

We always use PBS w/o Ca & Mg to wash our cell cultures prior to the addition of trypsin/EDTA. I remember one day many years ago one of the lab scientists just couldn't get their cells to lift. Had about 8 flasks sitting in the incubator and the cells just wouldn't budge. Turned out we'd received a shipment of PBS WITH Ca & Mg and nobody had noticed! You definitely need to use a Ca free buffer to wash your cells with when using trypsin/EDTA.

With regard to anti fungals we use fungizone at low concentration in all our media without harmful affect. It can be very nasty to cells at higher concentrations though. If you can get by without it I would. Our main problem is we can't afford to loose any of our primary cultures and about once every 12 months or so we'd get a low grade contaminant (usually yeast) in a bottle of media and it would cause all sorts of problems for us. We don't take the risk anymore.

I think the Trypsin:EDTA issue was well covered above, so I won't comment on it.

In regards to antifungals, we used to use fungizone all the time in our media. After about a month of unexpected results, however, we discovered that it actually induced expression of our protein of interest! So now, no more fungizone, but also no problems with contamination.

_Hank