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I am trying to develop a method for analysis of small cell numbers (<500),
which would allow us to analyse the contents of cell mixtures, and obtain
by image analysis, the relative fluorescence intensities of at least three
populations in the mixture:
Cells labelled positively for CDnn-FITC, nn being various marker numbers
Negative controls for the same antibody isotype
MESF-beads from FCSC, admixed.
Later on, we want to quantify the CDnn on the cells' surfaces.
We use Foster Findlay's PC_IMAGE software.
I have done this previously with Flow methods, where the cell numbers can
be much higher and where there is no bleaching of fluorescence.
In the image analysis setting, I am bedevilled by fading. What can I do? Do
the anti-fade agents work well, and which is best for human cells and mouse
monoclonals? Can I overcome the fading problem by minimising its effect
(set up on one field and measure on a fresh field at a set time post light
exposure), or by taking multiple image samples vs time and extrapolating
back,...??
I am also unsure if there may be a flaw in my approach of separately
staining the two cell populations, then mixing them just prior to mounting
a slide to make the measurement. My concern is that there may be
equilibration of CDnn-antibody between the newly added (negative control)
cells and those previously labelled as the test.
No, I don't have reasonable access to a Flow analyser.
I would like some of you image-experienced List observers (Al, Diether,..)
to point me in the right direction (by way of papers), and tell me of your
own methods that work.
My best wishes, and thanks to those who replied to my Anecdotal. I will be
responding soon, if you've not already heard from me.
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CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the
Purdue University Cytometry Laboratories and distributed free of charge
as an educational service to the cytometry community.
If you have any comments please direct them to
Dr. J. Paul Robinson, Professor & Director,
PUCL, Purdue University, West Lafayette, IN 47907.
Phone: (765)-494-0757;
FAX(765) 494-0517;
Web