9 replies to this topic

[crisyim]

Hey guys,

I have a problem regarding the harvesting of post-confluent adherent cells (Caco-2). I use TrypLE Express to harvest adherent cells but it doesn't work very well for post-confluent cells because I don't get single cells. First I wash the cells with PBS (without Ca and Mg). Subsequent I inkubate the cells with TrypLE for about 10 min at 37°C and knock the flask several times and/or try to seperate the cells with the pipet.
Has anyone of you an idea to get rid of this problem?

[DavNu]

you can try 1000 µl pipette tips but I will use accutase instead of trypsin works nicely and remaining clumps can be easily broken with p1000 up and down.

[bob1]

You may be trypsinising too long, this often causes cells to form inseparable clumps.  Why are you using post-confluent cells?  This alters gene expression and selects for a population of cells that are capable of surviving these conditions - changing the phenotype of the cells alters your experiments!

[leelee]

I agree with bob1

[CAT]

Agree! 10 minutes at 37C is too long. You may get some sticky lysed cells. You do not have to get all the cells. If cells still stick to flask, just leave them there, only collect those trypsinized cells for next passage.

[bob1]

CAT, on 29 March 2012 - 06:36 AM, said:

Agree! 10 minutes at 37C is too long. You may get some sticky lysed cells. You do not have to get all the cells. If cells still stick to flask, just leave them there, only collect those trypsinized cells for next passage.

No!, this selects for a population of cells that are weakly attached/more susceptible to trypsin, thereby changing the cell line.

[CAT]

Yes, it will if we are selecting tyrpsin-resistance or sensitive cells on purpose. There has reports about it too.  But here, he/she  already treated cells with trypsin for more than 10 minutes at 37C and no cell detached. Within the text, these post-confluent cells are "resistance" to trypsin, not because they changed, but some other reasons, like inaccessible to trypsin.

[crisyim]

Thank you for all the answers!

@bob1: I need konfluent Caco2 for a cell culture-based epithelial model (for this experiment I need the changed phenotype). But afterwards I like to analyse the cells by flow cytometry and therefor I need single cells.
I will try shorter trypsinising times.

@DavNu: I tried 1000 µl pipette tips but unfortunately a lot of cells cumps remain. But I will try accutase.

@CAT: Unfortunately I need (almost) all the cells for flow cytometry.

So, now back in the lab trying to implement your suggestions.

[Shadan]

iam having the same problem , when i triptinized for less time it didnt work
And more time just gave me ruined cells!!!

So , what happened with u Crisyim?

[Shadan]

Trypsinized*