
trypsinization of adherent cells
#1
Posted 28 March 2012 - 05:26 AM
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?
#2
Posted 28 March 2012 - 06:18 AM
#3
Posted 28 March 2012 - 12:24 PM
#4
Posted 28 March 2012 - 09:18 PM
#5
Posted 29 March 2012 - 06:36 AM
#6
Posted 29 March 2012 - 12:03 PM
No!, this selects for a population of cells that are weakly attached/more susceptible to trypsin, thereby changing the cell line.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.
#7
Posted 29 March 2012 - 01:07 PM
#8
Posted 30 March 2012 - 07:01 AM
@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.
#9
Posted 10 June 2012 - 01:44 PM
And more time just gave me ruined cells!!!
So , what happened with u Crisyim?
#10
Posted 10 June 2012 - 01:45 PM
Also tagged with one or more of these keywords: Trypsin, Caco-2, Adherent, postconfluent
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