RE: Alternative to BrdU labelling in vivo ?
kukuruga%kasle1.dnet@rocdec.roc.wayne.edu
Tue, 14 Mar 95 10:46:08 -0500
Response to Aki Hoji....any assay you employ will involve the use of H33342
in some way, as it is currently the only reliable way to determine cell
cycle viably and by flow. Its use requires several considerations:
1) UV excitation; and, since you want to use antibodies in combination,
usually a second laser (although there are UV excitable dyes available
which bind proteins and emit higher than H33342. You need to get creative)
2) As a vital label for DNA, obviously the cells are involved. You need
to balance dye concentration (usually 10uM works best) with cell number
(1-2 x 10e6/ml, NO MORE), and optimize time of incubation (at 37C) to
obtain good histograms with comparable stats to PI. Time can range from
30 to 120 minutes, and must be determined for each tissue type you use.
I have found, however, 10um for 1-2 x 10e6 cells/ml for 60 minutes in media
supplemented with 2%FCS (pH 7.2) works well for most cells.
You need to run a few pilot experiments to determine these conditions.
3) Keep in mind most cells will process surface antibody to some extent at
37C, so label with H33342 first.
4) Make sure you run controls...cells labeled with MoAb alone, H33342 alone,
etc.
A couple of other points...you don't need a lot of UV (I've achieved
PI-comparable results with 15-20mW), and this is best anyway, assuming you
want your cells to survive the process. (H33342, while a "vital" label,
does have mutagenic potential. This, in combination with the UV zap, and
anything is possible)
....This works well for Human cells. If you're talking rodent, things change
but only slightly. I usually reduce time and maybe dye concentration, but,
again, it needs to be optimized.
MAK.
CD ROM Vol 2 was produced by 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:(317) 494-0757; FAX (317) 494-0517; Web http://www.cyto.purdue.edu
EMAIL robinson@flowcyt.cyto.purdue.edu
