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1. "What is nonspecific binding?" How do you all out there control for it?
Isn't it in fact a less avid or FC binding? Are you all blocking the
appropriate FC receptors in your staining processes. It seems to me that if
you have blocked appropriately then there is no such thing as "nonspecific
binding" --- only unexplained phenomena.
2. I do samples from SCID mice engrafted with Human-PBL's from blood, spleen,
lymphnode and peritoneal washes. Quite frankly worrying about tightening a
"lymphocyte gate" by light scatter is superfluous for me because the mouse
cell light scatter gates do not coincide with human light scatter gates. Hence
I use flourescent vs side scatter (right angle scatter) gates to separate the
human from mouse cells. For example-- One sees those large CD3 positive
"lymphocytes" that have been reported as well as the regular population of CD
3 positive normal lymphocytes easily here, and can set a gate around these
"T-cells" to get a CD4/CD8 ratio directly from one tube. Also apparent are
those rare CD3+/CD4+/CD8+ cells that we all wonder about. Fortunately I don't
work clinical applications, but it seems to me that in light of modern
immunology that if we really believe this stuff, then the old light scatter
gates with their overlaps, need to be used in context to give us what we
really want. How many of you have looked at a CD3/CD4/CD8 stained whole blood
using flourescence vs sidescatter gates to see what you really have? This can
be really enlightening the first time you do it, especially if you have set a
"lymphocyte gate" by forward and side scatter gate under Cellquest with color
gating. Then paint your CD3's, CD4's and CD8's by setting gates on side
scatter vs flourescence. Finally change the priority (precedence) of the
gating and watch what happens. Then try and abnormal.... Have fun!!!!
My point is this -- as we move into a 3-4-and 5 color flourescent world, we
need to observe and be able to read patterns that give us meaningful
interpretations based on today's science.
Just as taxonimists used visible means to classify plants and animals and
interrelate them before we had DNA analysis, we used those tools available to
us in a 4 parameter, 2 color world for a long time to do the best job we
could. Maybe it's time to move way beyond this. In the transition, those of
us stuck with 3 color will have to make do,but wouldn't I like to run
CD3/CD4/CD8/CD14/CD45 all in the same tube on a machine that didn't cost
$250,000, require a full time operator to justify it, etc.? You bet I would.
In the meantime we all need to be as creative and imaginative as our current
instruments and software packages will let us be to push the boundaries of
science.
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