all my cell lines detached within five minute trypsin except one. i dont know which is better for cells and less harsh: to increase amount of trypsin from .7 to 1 ml for t-25 flask or to increase time from 5 to 8 min? i wash with dubelco PBS twice.
Does your trypsin have EDTA? Sometimes cells adhere to the plate and won't come off easily if there's no EDTA. The cells remain attached to the ECM via integrin receptors that require cations like calcium. EDTA is a chelator that binds those cations, so by adding it, the cells can detach faster.
Yes I agree. I increase the amount so it touch every single cell, but not increase the time not to increase the harmful effects .
I agree with Nandini, you should increase the amount and not the time. Also, I think that 0.7 ml is too little, I used to add 1.5 ml Trypsin-EDTA for T-25 flask.
I do have the same problem earlier. But i'd prefer gentle shake while incubating T-25 flask with tryp-edta. I used 0.3ml. it works fine.
It seems detachment time is also depends on specific cells lines. I worked with HepG2, HeLa, A431, MCF-7. HepG2 was once took even more than 5 min. but all the four were within 5 min with gentle shake while incubating at 37C.
Hope it help!
1. Which cell line and media are you using? Sometimes if you do not remove media completely from the wells, there are certain components in the media as well as the serum which inhibit trypsin activity. So make sure you remove the media properly.
2. Also some cells require longer incubation in trypsin. However, do not incubate longer than 10 minutes as it could damage the membrane proteins and kill the cells. Optimization of the incubation time is important in this regard.
3. You might also try other alternatives. You can increase EDTA concentration. Also you can try other commercially available reagents like TrypLE™ Select (1X) reagent, TrypLE™ Express reagent etc. Also you can try gentle scraping. However, this is not recommended for flow cytometry experiments. Read about the alternative procedures and reagents available for cell dissociation and optimize the condition according to the cell line and the experiment.
For T25, i normally add 1ml of 0.25% trypsin EDTA, for T75, i used 2ml trypsin. Please make sure u wash your cells with PBS prior trypsinization cause serum will inhibit tyrpsinization. But if the cells really hard to detach, i just use cell scraper.
I agree with Vabeiryureilai, the detachment of cells are cell type dependent. Some cell lines need lesser time and trypsin than others. If you wanna keep the cells in culture for many passages, it is recommended to use 0.5mMEDTA instead of Trypsin-EDTA, cause it can protect the cells normal karyotyping. EDTA will just losen the cell attachment to the plate surface but wont harm the cell-to cell attachment. While using EDTA, you need to incubate the cells for 10min and then remove the EDTA by aspiration, dont shake the plate during this time. Then put the medium ad remove the cells and do the following procedure. TrypLE is better than Trypsin and works similar to EDTA but very quick, most of time one minute is enough and then remove from the well and incubate the plate for 5-10min, then add medium and do proceed. Hope So, it can help.
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