It all depends on the cell line you are using. For example, if you are using Colon cancer cells for instance, those cells require much more time to detach. However, It also depends on the concentration of Trypsin you are using: 2 minutes if using 10X because leaving the cells bathed in 10X trypsin would provide a harsh condition if left there for more than 2 minutes.
What I advice you to do is to add 2 ml of 10X trypsin, incubate for 37 degrees, shake gently and observe under the microscope, if the cells have detached, neutralise the trypsin by adding 3-5 ml of media.
I prefer to work with 1X trypsin, too little cytotoxic effect is observed.
Good luck
We detach cells from plastic surface as follows:
- 1-2 ml/75 cm2 of 0.25% trypsin - 0.5 mM EDTA solution (Sigma-Aldrich Co.) (the quantity depends on the cell type we work with, some cells are more or less adherent)
- 3-7 min incubation at 37oC (the incubation time can be adjusted by contrast phase microscopy monitoring the amount of detached cells)
- enzymatic inactivation with 2-4 ml of fresh culture medium supplemented with FBS
- centrifugation
Although, there are cell types that hardly can detach from plastic surface using trypsin - EDTA treatment. In this cases you might eventually use a cell scrapper.
May I know how to remain Trypsin temperature under microscopy observation?
I would like to observe the characteristic of cell in remaining Trypsin temperature.
You might find useful a micro-CO2-incubator designed for use on the stage of an inverted microscope. These pieces are designed for real time observation of cell cultures.
the addition of EDTA-Trypsin and incubation even at room tempt. 5 min. will cause cell detach and might use scraper to speed up the detach.
Thank all.
May I know about microtissue detach? I used same method but I found out that EDTA-Trypsin temperature will reduce from 37 to 27 degree celsius within 3 minutes only. These period is not enough to separate all the cells.
37C/5mins, but depends largely on the brand/company. you have to standardize accordingly.
,i accept Gălăţeanu Bianca point . normally we used 500ul for 25cm2 flask (0.25%trypsin and 0.05mM EDTA)
37C for 2 min sufficient to detach the cells, however extend the time may required but take care of the toxic effect on cells.
it depends on the flask you were using, for T25 -1ml, T75-2-3ml, for 60mm n 100mm dishes it will vary, incubate it for 3-5 mins in incubator . based on the confluence if it is 90% even after trypsinization you can still find lot of cells in the flask jus tap the flask after incubation remaining cells will get detached.
you get ready made solutions of trypsin then what you want to optimize?????????? all the answers given were of standard protocols...
It all depends on the cell line you are using. For example, if you are using Colon cancer cells for instance, those cells require much more time to detach. However, It also depends on the concentration of Trypsin you are using: 2 minutes if using 10X because leaving the cells bathed in 10X trypsin would provide a harsh condition if left there for more than 2 minutes.
What I advice you to do is to add 2 ml of 10X trypsin, incubate for 37 degrees, shake gently and observe under the microscope, if the cells have detached, neutralise the trypsin by adding 3-5 ml of media.
I prefer to work with 1X trypsin, too little cytotoxic effect is observed.
Good luck
The effectiveness of Trypsin is very temperature dependent. Also, if Trypsin is too concentrated, it can damage cells. Leaving cells in 1X trypsin can also be harmful to cells so it is best not to compromise the integrity of the 95% detached cells for the remaining 5% cells still attached.
Keep the trypsin warm and only add it once you washed your cells. Then place it immediately in the warm incubator for 2minutes initially in 1X. Then tap the flask to see how detached your cells are. Then put flask back into the incubator for further incubation. Perhaps check every minute from here on. The key is to work very quickly so that the solution does not cool down too much.
When you wash your cells etc, have the flask resting on a a sheet of plastic for example so that the flask does not get too cool on the metal surface. Same goes for viewing under the microscope, that is work fast and only place the flask on the viewing platform when light is on etc.
Trypsinization of cell lines is temperature-dependent (as many have noted). Most researchers pre-warm the trypsin to 37 degrees C, but most incubate the cells in the trypsin at room temperature for 4-6 minutes during the digest. Your cell line may require different handling. Some prefer to return them to an incubator for that time and maintain the temp at 37 C.
I would also point out that primary cell lines (coming directly from the tissue, before 1st passage) can be very sensitive to trypsin or other similar enzymes. A lower concentration of 0.05% trypsin is often used for the first several passages to avoid damaging the cells (1X is generally 0.25%). Washing the cells with calcium & magnesium-free PBS can also help reduce cell attachment and makes it easier to get the cells to detach.
By using Trypsin-0.25% EDTA, on 37oC for 5-10 min. We use it on human cells and it works.
The incubation time is depend on your cell type. I think trypsin is not good for cells. Avoid prolong incubation more than 5 min. It is better to use mechanical methods.
I leave my cells (HeLa and Normal Human Dermal Fibroblast) for 3-4 minutes at room temperature (of course in the Biosafety Cabinet) once I have added my Trypsin
We tryed both ways:a) let the cells with trypsin in laminar box at RT; b) put the cells with trypsin back in incubator at 37°C.
Transfering the cells into the incubator can higher a risk of contamination, because you´re manipulating with culture vesels, taking them out of the laminar box. On the other hand, it also shortens the time you need to expose your cells to trypsin. And this is good, because longer exposure to trysin will damage you cells.
As said Mr. Nami, the incubation time depends on cell type, so you must check it every few minutes. Once the cells became round-shape, strike the culture vesel (we strike it /bank it against our hand), so the cells will be detached mechanically.
Good luck!
From my experience, using trypsin at room temperature usually works but for cells that are relatively difficult to trypsinize, I would pre-warm my trypsin at 37 degrees Celsius.
I do agree with Leo , but we warm trypsin at bit higher 40 degree and its working fine.
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