Hi Jim,

As others have indicated, these cells do take a really long time to trypsinize. But as ATCC recommends, it is advisable to use only 0.05% Trypsin. I have tries using 0.25% and the cells change their morphology and do not appear healthy. As others have suggested, wash these cells 2-3 times with 1XPBS prior to adding the trypsin and incubate them at 37. It takes anywhere between 20-30 minutes. Also, you can resuspend these cells after trypsinization in growth medium containing twice the required serum concentration. This effectively inhibits trypsin. You could then collect the cells, and resuspend in regular growth medium for culturing.

Regards

Pavan

Hi Jim, have you tried to wash them with trypsin, then remove most of it and let them detach at 37ºC? That's what I do for hard-to-detach cells. I remove medium, wash thoroughly with PBS and once with Trypsin. Then I leave 2.5 ml Trypsin in a 15 cm dish and put it at 37ºC for 5 to 10 minutes. I think that confluence is sometimes directly related to the difficulty to detach. Hope it helps!

Hi Jim,

I used currently these cells and 10-15 minutes is necessary but if I add directly the media in the plate to neutralize the trypsin it is impossible to detach. So detach it directly in trypsin and add immediatly the cell in a 15 ml tube with media+FBS.

These cells do take long to trypsinize. After 5-10 min, I use a 1 ml pipette to suck the trypsin up and squirt it on the cells to try to wash them off the plate with the trypsin. This helps them come off faster. In our lab, we 'quench' the trypsin with a high-serum media and then centrifuge the cells at 900 rpm for 3 min to recover the cells from this medium. We resuspend the cells in the growth media for plating and passaging - it is important to remove the trypsin from the cells by centrifugation, because over time they will become more mesenchymal in the presence of residual trypsin.

I wash them with PBS and then leave them with trypsin for 20-25 minutes. You will get most of them but It is really hard to get all of them.

Wash with PBS, add trypsin and incubate at 37 degrees in co2 incubator.Incubating at 37 should help in case you did not do that.

Most important thing is to make sure that your trypsin is stored properly and is active.If you are using inactive trypsin to detach your cells obviusly you will have problems detaching your cells.

Thanks for the advice! Much appreciated!

Jim

I wash the cells with warm PBS pH 7.4 throughlly and the add 1.5 ml of trypsin EDTA incubate for 2 minutes. My cells get separated very well. I never had any problem like you did. Good luck

Sunny

http://www.ncbi.nlm.nih.gov/pubmed/12798140

This is a detailed methods paper on the culturing of MCF10a.

I have never used MCF-10A cell lines. However I have some experience with cells that attach so strongly to plate. For those I use 0.25% Trypsin to briefly wash and the same solution to detach them at 37 C, typically 10 min or less incubation. You should be careful not have the thrypsin around your cells for too long. Of course as soon as the incubation ends, the trypsin should be neutralized with FBS. Good luck!

Dear Jim,

Agree with all, because for "srong sticking cells", trypsin has to be the most efficient as possible. And often some compounds of your currrent culture medium can "neutralize" trypsin. And to be sure to remove all medium before action, washing cells with PBS is good. Also, but I suppose verified by you, when trypsin is de-frozen and reconstituted to act, once it is at room temperature (or 37°C), it auto-digests it self; so, activity can decrease regularly according to time.

Regards

Didier

Hi Jim,

As others have indicated, these cells do take a really long time to trypsinize. But as ATCC recommends, it is advisable to use only 0.05% Trypsin. I have tries using 0.25% and the cells change their morphology and do not appear healthy. As others have suggested, wash these cells 2-3 times with 1XPBS prior to adding the trypsin and incubate them at 37. It takes anywhere between 20-30 minutes. Also, you can resuspend these cells after trypsinization in growth medium containing twice the required serum concentration. This effectively inhibits trypsin. You could then collect the cells, and resuspend in regular growth medium for culturing.

Regards

Pavan

As most people mentioned, use PBS (without Ca+ and Mgcl2) wash before trypsinizetion. You may try 1x TrypLE Express up to 30' (its a trypsin-like recombinant enzyme; RT stable; can be substituted for trypsin; Even you can use PBS dilute to inactivates it).

first remove the spent medium and wash the cells once with PBS (approx 10 ml). This should be followed by trypsin-EDTA for 5 minat 37C in the CO2 incubator. Since these cells attach very strongly, you need to tap them at the sides (all sides) a bit more strongly than you do for other cells. If you are still finding many cells attached Incubate for 5 more minutes and try using CELL STRAINER. THIS SHOULD HELP YOU TO COMPLETELY DETACH ALL THE CELLS from the flask.

All the best and do let me know if you need additional help.

To complete Pradeep advice, and using Trypsin-EDTA mixture, evidently better than trypsin allone (EDTA calcium chelator) , and also if your cells are sufficently "solid", you can complete in the trypsin incubation period with a manual action: scratch gently with a rubber policeman or , has said previously, pipeting-rejecting the medium several times with a 1ml-pipetman.

Regards

Didier

I have no experience in culturing MCF-10A cell lines. Many cancer cells I have used can be detached by adding 0.25% trypsin and incubating at 37 degrees in co2 incubator 5 min after washing with PBS. You may change your Trypsin solution perhaps it does not work well.

All the best!

I am sorry, it is not CELL STRAINER, BUT CELL SCRAPPER. All the very best. Donot use Trypsin, Instead use TRypsin-EDTA as suggested by didier

Hi Jim,

I have no problems in trypsinize the MCF-10A cells. Try Pipette out the trypsin in the flask after 5 minutes icubation follow by adding in fresh trypsin. It might be the left over of Horse serum which inhibits the trypsin activity. Hope this works for you. Cheers

Hi Jim,

Try giving a wash with 0.53M EDTA after the PBS wash. Then add Trypsin-EDTA and see the detachment. Atleast I have tried it for the cells that adhere very strong and suceeded...! Good luck :)

A lot of great advice! Thanks again everyone!

Jim

If they are sensitive a little bit of PBS for a few mins might be enough to dislodge the cells with a strong whack.

Hi, just wonder can we mix TrypLE express (0.05% Trypsin 0.53 mM EDTA) and normal Trypsin-EDTA (EDTA (0.25% Trypsin, 1 mM EDTA.4Na), for example 1:1 ratio? Since TrypLe is hard to detach cells....