Fwd: Another Acridine Orange Question

thomas.delohery@VERIZON.NET thomas.delohery at VERIZON.NET
Tue Jun 8 21:58:25 EST 2004

gee, I might be able to help with this one.  Please see answers below. 
 Feel free to contact me directly if you have any other questions. 
 There are numerous other caveats I would be happy to share.  

good luck, Tom Delohery, writing from home....

Thomas Delohery, Principal Scientist
Cellular Immunology, Immunology Platform
Aventis Pharmaceuticals
thomas.delohery at aventis.com
1041 Rt. 202-206, Mailstop BWG-303
Bridgewater, NJ    08807

Jeanene Pihkala wrote:

> Subject:
> Another Acridine Orange Question
> From:
> "Jeanene Pihkala" <jpihkala at mail.mcg.edu>
> Date:
> Mon, 07 Jun 2004 17:36:52 -0400
> To:
> <cytometry at flowcyt.cyto.purdue.edu>
>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
>1- Since AO is so bright should it be run on lin or is log okay?
We acquired all data with linear amplification because accurate ratio 
measurements require linear fluorescence measurements when using analog 
signal processing electronics. We were using a FACSCalibur at the time.  
I am curious about ratio calculations on a digital system in case anyone 
wants to chime in.  We used FlowJo to calculate Red (>650nm): Green 
(510-530nm) post-acquisition.  You have identified the major obstacle in 
measuring AO fluorescence in a linear scale - "brightness" or 
fluorescence intensity.  I was fortunate enough to work with an 
individual (Shoshana) that was compulsive about growth and labeling 
conditions used in the experiments.  Even so, keeping the cells on-scale 
with linear amplification can be problematic.  Shoshana worked out the 
labeling conditions so we could reproducibly keep approx. 90% of the 
cells on scale.

>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? 
For our system, the green fluorescence changed very little between 
controls and treated (irradiated) samples.  The formation of acidic 
vessicles causes a huge increase in red fluorescence.  Green 
fluorescence did increase in the treated samples but nothing in 
comparison to the red.	So we would set up the instrument with the 
untreated controls such that the red and green were approximately equal 
and as low on the linear scale as possible.  This yields a Red:Green of 
approx. 1 and allows room for the red fluorescence to increase in the 
treated samples.  From experiment to experiment, the instrument set up 
is an iterative process of running the controls and the treated samples 
and adjusting the PMT voltage until you get  approx. >90% of the cells 
on scale - for both the controls and the treated samples.  If this can 
not be accomplished you need to adjust the labelling conditions.  At 
least the "90% on-scale" was the criteria we felt comfortable with for 
the cell lines.  You may need to set a different criteria if you are 
looking at primary cells.  

>Thanks in advance for any help anyone can offer.
>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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