Wash cells or not after trypsinization? - (May/03/2005 )
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!
)Hi It Abhi
If you are using synthetic media in which you don't add serum (minimal medium) the it is a must to spin down and wash the cells with cold PBS after trypsinisation. But when an enriched media is used containing 5% FBS then it is not necessary to wash because the serum has alpha antitrypsin activity in it
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!
)Hi,
If you are trypsinizing cells for regular maintenance, you need not wash cells but if you are going to use trypsinized cells for some assay, it's a good cell culture practice to wash your cells. You never know what will change your results in cell based assays, better to minimize such kind of variables.
Hope it helps.
Pravin Mahajan
Other enzymes which are less harsh can be used as an alternative to Trypsin, causing less damage to the cells. In general you would use these on primary's as most cell lines are fairly resistant to trypsin. They are all inactivated in the same way as trypsin, using FCS/FBS. These include :-
Collagenase's : Types I, II, III and IV.
Elastase
Hyaluronidase
Dispase
If you are having trouble with your cells and you think it is the trpysinisation which is causing the problem, try an alternative.
Our lab uses 'Citric Saline' which is made inhouse and composed of simple salts. It works well (similar to trypsin) for some cell lines.
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!
)if there are running protocols in a lab, or if you strongly refer your work to published results, methods should be adopted including inhibition of trypsin with medium or not, so that protocols and results remain comparable; in general, I agree that primary cells are more sensitive to trypsin than cell lines cells
We don't wash after trypsinization, but remove the trypsin and then add the medium just to be sure.
When I use trypsin without EDTA, I always add medium contains FBS after trypsinization. It works well.
But when I use trypsin with 0.02%EDTA, I wash after trypsinization. My colleagues tell me that can get rid of the EDTA.
I know EDTA can combine with Ca2+ etc.
I wonder there are any more disadvantages when cells are cultured in media contains EDTA?
Dear All,
I cannot believe this thread is still running. Up until this thread started, I had never heard or met anybody who washes their cells after trypsinisation. I have cultured both primary's and cell lines over 30 years and always used the standard method i.e. adding media ( Ca2+/Mg2+ and protiens) to dilute and inactivate the trypsin, centrifuging, removing resultant old media, resuspending and plating out.
I always prewarm the trypsin and trypsinise at Room Temperature for 30 seconds.
Some cells are more resistant so wash x3 with PBS (- Ca2+/Mg2+) and then wash x1 with EDTA (Versene).
As stated in a previous post, Elastase, Collagenase, Hyluronidase, Pronase etc can be used as an alternative.
From the voting NOT WASHING is the majority view.
Trypsin can be inactivated by cation such as Mg2+, Ca2+which these cation have enrich in serum. When you add media contain serum, Trypsin also inactivate.
500 ul (0.25mg/ml) is use for 75cm2 flask.
1,5ml of trypsin is enought for the T75... 0,5ml for T25 and so on
