Apoptosis among CD34 positive cells
Norman B. Levy (Norman.B.Levy@Dartmouth.EDU)
26 May 95 17:57:27 EDT
We did a number of experiments a few years ago looking for a "universal"
fixation procedure for performing surface, cytoplasmic and nuclear stains by
flow. We ended up with a protocol adapted from Raul Braylan. Basically we
stained the surface of unfixed cells, then used a SHORT 2 min. exposure to 1%
paraformaldehyde in PBS to fix the antibodies to the surface before
permeabilizing with ethanol/Triton X 100. During the course of these studies we
noted that ethanol had the impressive ability to knock off phycoerthrin and APC
from the bound antibody. Further, this effect appeared to be antibody and
manufacturer variable. In other words Coulter's method of conjugating PE to
antibodies appeared much more susceptible to this alcohol effect than B.D's
method, but for some CD's we never found an acceptable PE labelled antibody. We
tested a large number of antibodies, but never tested CD34. Interestingly, the
alcohol effect was not as bad for FITC. If you can get enough signal, you might
try a FITC labelled CD34.
Alternatively, you might want to evaluate other permeabilizing protocols that
avoid alcohols. The paraformaldehyde trick of fixing the antibodies to the
surface before permeabilization should be retained regardless of subsequent
fixation. The exposure has to be short however, because prolonged exposure
permeabilizes the cell and cross links internal proteins and DNA and mucks up
PI staining of DNA.
We tried to publish our experience, but the bad boys in Cytometry didn't like
us.
Norman Levy,
Dartmouth Hitchcock Medical Center
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
