RE: FISH on sorted myeloid progenitors

Hi

I dont have experience with FISH, but most fluorescence microscopy uses a fairly broad excitation source, so I expect APC will show up in a set-up for Texas Red.  But its easy to check - they should do a sort and then put the cells on the microscope and see if there really is appreciable signal.  If there is, they might be able to treat the cells with pronase or another protease to cleave the Abs from the cell surface






=======================================================
Rachel M. Gerstein, Ph.D.
Associate Professor
Department of Molecular Genetics and Microbiology
Graduate Program in Immunology/Virology
University of Massachusetts Medical School
55 Lake Avenue North
Worcester, MA 01655-0002
(508) 856-1044
(508) 856-5920 (FAX) 



-----Original Message-----
From: Avery,Anne [mailto:Anne.Avery@ColoState.EDU]
Sent: Wed 4/9/2008 3:03 PM
To: cyto-inbox
Subject: FISH on sorted myeloid progenitors
 
Hello Everyone,

I have a colleague who would like to use a Texas Red probe for FISH on sorted common
myeloid progenitors from irradiated mice.  I was hoping if someone could point out any
obvious flaws in our choice of fluorochromes, since I have no experience with this.  We
have a MoFlo with a 488, 635 and 405 lasers and hope to sort cells first and then do
FISH.

Outgate on lineage markers
	biotin conjugated CD3/CD19/Gr1/Ter119, streptavidin-PE

Various populations will then be positively selected using the following:
FcR-APC
CD34-F
Sca-Pacific Blue
c-kit-APC-Cy7

I was assuming that PE might interfere with seeing the Texas Red probe, so left it out of
the positive selection mix.

Thanks,

Anne




Anne Avery, VMD, PhD
1619 Campus Delivery
Dept. Microbiology, Immunology and Pathology
Colorado State University
Fort Collins, CO 805023
Ph: 970-491-1170/6138
Fax: 970-491-0603