Another Acridine Orange Question

Jeanene Pihkala jpihkala at mail.mcg.edu
Mon Jun 7 16:36:52 EST 2004


Hello Flowers,	

I have a user who is trying to duplicate an experiment from Cancer
Research, Vol 61, Jan 2001 [("A Novel Response of Cancer Cells to
Radiation Involves Autophagy and Formation of Acidic Vesicles" Shoshana
Paglin, Timothy Hollister, Thomas Delohery, Nadia Hackett, Melissa
McMahill, Eleana Sphicas, Diane Domingo and Joachim Yahalom)I've
attached a PDF of the article].   I need help from anyone who has done
this or has any ideas on the best way to do carry out this experiment.	

What the researcher wants to do is stain irradiated cells with AO and
look at green (510-530nm) vs. red (>650nm) fluor compared with AO
stained unirradiated control cells - this information is then displayed
as a ratio  of mean red:green fluorescence on a histogram. The questions
I have of the procedure that are not addressed in the journal article
are:

1- Since AO is so bright should it be run on lin or is log okay?

2- To set up the instrument I assume I set both red and green fluor at
the same channel intensity since on the graph of the ratio it shows a
baseline ratio of one; but where on the fluor scale would be the most
appropriate place to set them and what type of sample should I use to
set them with since there really is no red only and green only? 

Thanks in advance for any help anyone can offer.

Jeanene

Jeanene Pihkala
Manager, Flow Cytometry Core Facility
Institute of Molecular Medicine and Genetics
Medical College of Georgia
1120 15th Street, CA2022
Augusta, GA  30912-2600



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