Re: Embryonic Mouse Brain sort

Dear Janet,

the "debris" are myelin/fat vakuoles from the brain. The only way to get a bit
rid of it is a good percoll densitiy separation. Always use 30 um filters
before sorting samples. Be not disappointed, there are few cells in the brain -
most part is fat/myelin. You can perfectly separate the cells when you include a
Hoechst/PI combination for myelin/dead versus live cell discrimination.
I will attach you a sample from human brain (Hoechst x axis, PJ y axis), it is
similar to mouse brain. But the amount of myelin varies considerably if you
take lateral ventricle, cortex etc.

Hope that helps.

Best regards

Conny




Dr. Cornelia Brendel
Klinik für Hämatologie, Onkologie, Immunologie

Universitätsklinikum Gießen und Marburg GmbH,
Standort Marburg
Baldingerstraße
D-35043 Marburg

Telefon   (+49) 06421 - 286 5061
Telefax   (+49) 06421 - 286 5062
E-Mail: Brendelc@mailer.uni-marburg.de
url: http://www.med.uni-marburg.de

Aufsichtsratsvorsitzender: Wolfgang Pföhler
Geschäftsführung: Gerald Meder (Vors.), Prof. Dr. Werner Seeger (stv. Vors.),
Manfred Wiehl, Dr. Peter Mein, Prof. Dr. Martin Hansis (stv.), Dr. Hans-Jürgen
Hackenberg (stv.)
Sitz der Gesellschaft: Gießen
Amtsgericht Gießen HRB 6384
Email: BrendelC@mailer.uni-marburg.de




Zitat von janet dow <jldow@unity.ncsu.edu>:

> Dear Fellow FLOWers:
>
> I have a client who comes to me to sort disassociated embryonic mouse
> brains that have been transfected with GFP.  I am having real
> problems with the sorts as the samples contain large amounts of
> cellular debris as a result of the disassociation process. The client
> washes the samples multiple times trying to get rid of this debris
> with no success.
>
> As a result, my sorter lines are being clogged, and I must stop the
> sort multiple times to backflush the sample lines(this last sort I
> had to stop 10 times).	I am certain the debris is causing the cells
> to clump and/or stick to the sample tubing.
>
> I would be grateful to hear from anyone who may be doing similar
> samples with suggestions as to how we might stop this
> clumping/stickiness from disrupting the sort.
>
> I am using a MoFlo with a 100 um tip running at 30 psi and am using a
> SmartSampler.  My stream stays remarkable stable during the sort, so
> I am thinking it is the sample tubing that is being clogged.	The
> sample is put thru a 35um mesh before sorting to eliminate large
> clumps.  The sample is kept at cold during the sort.
>
> Thank you in advance for all the advice.
>
> Sincerely,
>
> Janet Dow
> --
> Janet Dow
> Research Specialist and Manager
> Flow Cytometry and Cell Sorting  Facility
> North Carolina State College of Veterinary Medicine
> 4700 Hillsborough Street
> Room C-309
> Raleigh, NC 27606
> (919)513-6443
>


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