What if you added some BSA, or maybe some DNase, or something to the solution to prevent clumping. The trypsin worried me for flow antigens as I know it cleaves VEGF receptors.
-----Original Message-----
From: Mark Tarango
Date: Wed, 09 Jul 2008 04:21:07
To:
Cc: ; ; ;
Subject: Re: [Histonet] Single-cell susension from tissue
That's what I've seen most people do when they re-clump. If you see debris
everywhere on the side scatter vs forward scatter histogram, you might add
some extra washes or digest longer to clear it up too. Cytometers are made
for analyzing blood/BM and if there are clumps you can clog it up. Careful!
On 7/8/08, Stephen Hyter wrote:
> thanks for the replies
>
> after much trial and error I am using a mixture of Collagenase IV, Dispase
> II and a touch of trypsin. It has actually worked pretty good, I should of
> mentioned I passed through a 70 micron filter but you could visualize clumps
> of cells in the suspension cter on
>
> I am thinking I will try pipetting them a couple of times before I pass
> through the filter, that should help right?
>
> stephen
>
>
> --- On Tue, 7/8/08, anh2006@med.cornell.edu
> wrote:
>
> > From: anh2006@med.cornell.edu
> > Subject: Re: [Histonet] Single-cell susension from tissue
> > To: "Mark Tarango" ,
> histonet-bounces@lists.utsouthwestern.edu, hyters@onid.orst.edu
> > Cc: histonet@lists.utsouthwestern.edu
> > Date: Tuesday, July 8, 2008, 9:06 PM
> > What enzymes are you using for digestion? Just curious ....
> > Have you looked into the BD Medimachines for cell
> > dissociation? They work great .... So easy!
> >
> > -----Original Message-----
> > From: Mark Tarango
> >
> > Date: Tue, 08 Jul 2008 20:43:24
> > To:
> > Cc:
> > Subject: Re: [Histonet] Single-cell susension from tissue
> >
> >
> > Hi Stephen
> >
> > You can vortex but I'd keep them in the enzyme bath
> > longer. If you're
> > worried about losing a population of cells (or the epitopes
> > getting chewed
> > up from digestion), remove the cells that have come out of
> > the tissue and
> > hold them in PBS. You can put the remaining tissue back
> > for digestion (you
> > don't want to exclude a population of cells because
> > they never came out of
> > the tissue). Different populations of cells can come out
> > at different times
> > and you can even end up selecting out populations based on
> > how fast you are
> > centrifuging.
> >
> > Mark
> >
> > On Tue, Jul 8, 2008 at 7:50 PM,
> > wrote:
> >
> > > hello, having trouble fully dissociating dermal tissue
> > to use in a cell
> > > count on a hemacytometer. Can I vortex cells in
> > suspension or should I just
> > > keep them in the enzyme bath a bit longer? (worried
> > about surface markers
> > > for FACS)
> > >
> > > thanks for any help,
> > > stephen
> > >
> > > _______________________________________________
> > > Histonet mailing list
> > > Histonet@lists.utsouthwestern.edu
> > >
> > http://lists.utsouthwestern.edu/mailman/listinfo/histonet
> > >
> > _______________________________________________
> > Histonet mailing list
> > Histonet@lists.utsouthwestern.edu
> > http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>
>
>
>
|