(by nobody from nospam.not)
Wed Jul 9 05:39:33 EST 2008
dk from no.email.thankstospam.net (DK) wrote in
news:BcXck.350$BC4.114 from newsfe07.lga:
> In article
> <df5cb6d2-51fc-4e3a-839b-83b41681af24 from d1g2000hsg.googlegroups.com>,
> Lara <eudolin from googlemail.com> wrote:
>>Hello there folks,
>>I use for some endothelial cells trpsin/edta and according to the
>>protocol I have to pipette in 5 ml trypsin/edta and then immediately
>>remove 4.5 ml
> Not immediately. Slosh the dish few times and *then* immediately
>>and leave the rest for a couple of minutes. Does anyone
>>know why? Isn't this simply a waste of trypsin?
> No, it's not. There is always some medium left - almost 0.5 ml
> from 10 cm dish, typically. So it makes a huge difference
> whether you dilute it with 0.5 ml or 5 ml of trypsin/EDTA.
> Remember that most media contain serum and serum contains
> powerful trypsin inhibitor - you want to add enough trypsin to
> titrate it out. Likewise, medium contains Mg and Ca - you want
> to add enough EDTA to chelate them or cells won't detach.
> You could just wash with EDTA and add little trypsin but the
> cost of doing in sterile pipettes and time exceeds the cost of
> the crude trypsin preparation used in cell culture.
>>Furthermore, is there any place where I can find detailed info about
>>trypsin in particular? Thanks for any info.
> Any of the hundreds of the cell culture practical guide books.
> Some cell lined don't even require trypsin - just EDTA is
I agree with DK.
On the other hand, we generally use collagenase, a rather drude
preparation with trypsin as a major contaminant. Because EC are generall
grown on a gelatin coating, you'll need a bit more enzyme activity than
when detaching cell lines from just plastic. It may even depend on how
you lay down the gelatin, coat 1 hr at RT, vs overnight at 4C.
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