[Cytometry] Compensation with liver cells
bestv at ccri.net
Fri Jul 10 15:03:57 EDT 2009
I agree problem is due to autofluorescence. I look at AF cells from liver
and other tissues, my isolation procedure is the same.
Would advise you to trust your beads over cell calculations, particularly if
AF cell populations are new to you. Of course the primary rule remains-
they need to be at least as bright as the fluorescence of your cell samples.
Which brings me to another point-
if I weren¹t already a bead user, given the recent posts, I might be afraid
to use them. No offense anyone, I have certainly enjoyed the discussion!
The main problem of the original inquiry is probably not the beads, it¹s the
autofluorescence and how best to compensate given this circumstance. And
there are many options for dealing with autofluorescence.
My approach is complicated, I use beads AND cells (need to discriminate
autofluorescence, need to make sure beads are as bright as cells, need to
see pure fluorescence without the autofluorescence), but it isn¹t the only
approach. But the fact remains that beads are easy and they do their job.
Not a politician and Thanks for the discussion,
On 7/9/09 11:30 AM, "Chaudhary, Kunal" <kchaudhary at ukaachen.de> wrote:
> Dear fellows,
> I have a big problem regarding compensation. I did compensation with beads
> which was accurate but as soon as i tried to compensate with liver cells its
> shows huge difference. I dont know which compensation set up to take and which
> not. I isolate the liver cells by perfusion (Collagenase type 1 + Dnase I) and
> than digestion again with collagenase + DNase I at 37 degree. Finally i
> measure my cells and i see again wired results. I didnt add any Ab labelled
> with percpe Cy7 and APC but still i get strong signals. I rechecked the PMT
> and compensations and all but i still cannot understand from where i get these
> signals. Please please help me.
> Thank you very much in advance,
> With regards,
> Kunal Chaudhary
> PhD student
> Department of Medicine III
> University Hospital Aachen
> Aachen, GERMANY
> Tel: +49-241-80-89200
> Cytometry mailing list
> Cytometry at lists.purdue.edu
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