Wash cells or not after trypsinization? - (May/03/2005 )

Hey,
I have had a bit of a discussion today, as to whether you will have to spin down and wash cells after trypsinisation or just add medium to the cell / trypsin suspension. I personally have never had any problems by just inactivating the trypsin by adding fresh medium, but would like to hear
your opinions too , before admitting defeat ( TA is always right! )

I previously washed the cells after trypsinization. After seeing a colleague omit the wash step, I adopted his method. So far so good. One thing I keep in mind is to minimize the amount of trypsin used such as using no more than 3 ml trypsin for a 75 flask.

I don't wash cells after tripsinization. I agree with use the minimun amount of tripsine, and i use it 1.5 ml tripsine 0.125% for a 100 mm petri dish with confluent fibroblast. Then neutralize with 5.5 ml of medium. After this, i take no more than 1 ml for subculture, so the amount of tripsine is minimum in the final culture.

I do both depending on how much trypsin is needed and if I have enough cells to dilute out the trypsin enough into media and still maintain the culture at the confluence I need. As long as you dilute out the trypsin with serum containing media they will be fine.

Hi,

I never wash after trypsin and i agree with the above, minimise the amount - i use 3ml in a T175 and inactivate by adding 7ml medium

hi
i wash cells only if they attach slight to the plate in order to enhance the reattachment step.
but for "classical cells" Hek HeLa, i don not wash.

But, what's the advantage if you omit the washing step? You save... like 5-10 min?

I only wash after tryp. for cells that are very sensitive to tryp like primary epithelial cells. Those cells will not reattach to the plate if you don't do one wash step with PBS. Cell lines like 293--never ever wash with tryp and they are fine.

With my primary cells I find that I get more live cells, as there seems to be some shearing going on when I spin them down.

Hi,

I always remove the trypsin after ensuring that the cells have detached; then add the medium to aspirate & re-plate. This way you don't have to worry about the amount of trypsin in the newly-plate cells.