Neeru: A better way at looking at DNA fragmentation in sperm is using Acridine Orange, which when bound to single stranded (denatured) DNA fluoresces red (> 630nm) when excited by a 488nm laser and fluoresces green (515-530nm)when intercalated in normal double stranded DNA. The following publications should be helpful in providing the assay details which we have found to work quite well. 1) "Sperm chromatin structure assay parameters as predictors of failed pregnancy following assisted reproductive techniques" Larson, DeJonge, Barnes, Jost, and Evenson Human Reproduction Vol. 15 (8)2000; pp1717-1722 2) "Sperm Chromatin Structure Assay for Fertility Assessment" D Evenson and L Jorst in Current Protocols in Cytometry (2000) 7.13.1-7.13.27. You may need to get some offline analysis software that can do parameter ration analyses to look at the ratio of Green:red. Joanne Lannigan, MS Director, Flow Cytometry Core Facility University of Virginia Jordan Hall, Room 7067 P.O. Box 800734 Charlottesville, VA 22908-0734 Office: 434-924-0274 Lab: 434-243-2695 Fax: 434-982-1071 email: joannelannigan@virginia.edu > -----Original Message----- > From: neeru sahni [mailto:neerums@hotmail.com] > Sent: Friday, January 23, 2004 3:31 PM > To: Cytometry Mailing List > Subject: Dna fragmentation in sperm cells > > sorry my attachments didn't went through last time so, i am resending this > querry from one of our customers > Dear flowers > I am studying DNA fragmentation in mouse sperm cells obtained from > Epidydymus. The cells were exposed to different doses of gamma radiation > like reported dose previously (10 Gy), stained with propidium iodide and > then acquired with FACScan. > The protocol for staining, instrument settings and the data obtained is > attached for your review. It will be of immense help if you can suggest me > the points that I might be ignoring in the whole process i.e. in staining, > acquisition or analysis of my data. > As you can see from my data that there is one prominent shoulder in the > irradiated sample, I am not sure about that how to interpret that as it is > within the haploid peak so I didn't count that as fragmented DNA. > I really appreciate it if you would give me some idea to solve it. > Regards. > Cengiz Yildiz > email:cengiz.yildiz@sickkids.ca > or > neerums@hotmail.com > > > > Instrument settings facscan > FSC E00 8.00 LIN > SSC 221 3.98 LOG > FL2 620 1.00 LIN > FL2A 1.00 LIN > FL2W 2.08 LIN > > _________________________________________________________________ > NRI's, Free Money transfer to India. > http://server1.msn.co.in/msnleads/citibankrca/citibankrca2.asp?type=hott ag > Click here.Received on Fri Jan 30 22:38:00 2004
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