How does it vary with different cell types? If I keep the cells longer in trypsin, what damage can be caused to the cells? What is the minimum and maximum time one can safely keep the cells in trypsin before adding media for neutralisation?
Trypsin acts by cutting amino acids, specifically lysines or arginies, unless these amino acids are followed by an proline. In cell culture, the enzyme is cutting away the focal adhesions that are anchoring the cell to the culture dish. Most trypsin solutions for cell culture also contain EDTA which acts as a chelator for calcium and it is important to rdeuce of trypsin-time incubation. You could use also only EDTA which is more soft but it needs a long time of action. The reasons for the range of time are as follows: (1) Differences in trypsin activity or potency; (2) Different concentration; (3) Different cell lines. Personally, I am using 2-3min at 37 ° for primary cells.
Incubating cells with too high a trypsin concentration for too long a time period will damage cell membranes and kill the cells. If unsure about the concentration of trypsin to use, use a low concentration. There can be lot-to-lot variation in dissociation times which is to be expected since the enzymatic activity of each lot will differ. If trypsin is being solubilized or diluted from a concentrated solution, it is important to use a buffered salt solution that contains no Mg2+/Ca2+.
Trypsin acts by cutting amino acids, specifically lysines or arginies, unless these amino acids are followed by an proline. In cell culture, the enzyme is cutting away the focal adhesions that are anchoring the cell to the culture dish. Most trypsin solutions for cell culture also contain EDTA which acts as a chelator for calcium and it is important to rdeuce of trypsin-time incubation. You could use also only EDTA which is more soft but it needs a long time of action. The reasons for the range of time are as follows: (1) Differences in trypsin activity or potency; (2) Different concentration; (3) Different cell lines. Personally, I am using 2-3min at 37 ° for primary cells.
Incubating cells with too high a trypsin concentration for too long a time period will damage cell membranes and kill the cells. If unsure about the concentration of trypsin to use, use a low concentration. There can be lot-to-lot variation in dissociation times which is to be expected since the enzymatic activity of each lot will differ. If trypsin is being solubilized or diluted from a concentrated solution, it is important to use a buffered salt solution that contains no Mg2+/Ca2+.
What cell line are you using right now?
It depends on the volume of trypsin you use also. With T25 flask, I use 1 ml, T75 flask 2ml, 100mm dish: 1.5 ml.
For HUVEC cell line: I only incubate 30-40 second
For HEK293T cell line: incubate 1 min
For NIH3T3, COS7, Hela,etc: incubate 1-2 mins depend on the cell density.
It is easy to detect whether the incubation time is enough or not by observing the moving of cells. When the cells are detach the surface, cells will move. You can tap on to the flask or disk to make the cell separate each other.
Thanks for your answer Trang. I also use different volume and concentration of Trypsin depending on the cell type and the culture plate. For hamster cells, 30 seconds of 500 uL of 20% Trypsin is enough for detachment. But for U2OS cells I have seen, at times some cells start floating much earlier, while others remain firmly attached as seen under light microscope. So then if I incubate more, will it damage the cells which are already detached and floating? What should I do then?
Tias,
You should incubate more until 80-90% cell detach from the surface. You can observe some cells detach sooner than the others (maybe 1-4%), these cells could be death cells or sick cells. By centrifuge steps, the dying cells will removed.
After the cells are trypsinized and detached the surface, you neutralize the cell with media containg 10%FBS. Then you centrifugue for 1min at 2,000rpm depending on cell type. I use this condition for NIH3T3, HEK293T, Hela, COS7 cell lines. You remove the media, the survived cells will in the bottom of falcon tube. Then you can wash with DPBS and centrifuge the cells. Then you add the new media into the tube and put them into new flask or dish.
The dead cells, sick cells, or debris are lighter than healthy cells. With low centrifuge speed, you can remove it.
It will have low ability that the dead cells or debris in the pellet. However, it will float during the incubation step. You remove it by washing step.
The main point is you have to incubate trysin until 80-90% cells detach from the surface. I hope it helps.
Just add 200-300ul for 25cm2 flask when 80-90% confluent and 500ul -600ul for 75/125 cm2 culturen flask. Keep it in incubatôr for 3-4 minutes in case of some cells. Hela cells and h292 cells are kept only for 2 minutes as they trypsinised fast. Most of the cells take 10-15 minutes in case of Hacat cells, hard cell lines. After getting out from incubatôr , tap the flask on hands to get cells from flask surface and check under microscope . And immediately pour complete media to dilute trypsin and nullify it damaging effect.
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