Fixation ofhuman lymphocytes
Antony Bakke (bakkea@ccmail.ohsu.edu)
Wed, 11 Jan 95 11:39:54 PST
In answer to your question about fixation of lymphocytes. Most
cell surface proteins will lose their antigenicity when fixed
with ethanol or methanol, but some can survive this. You will
have to experiment and compare fixed and unfixed fluorescence.
To prevent clumping during fixation with alcohols, resuspend the
cells in a small volume of saline (e.g. 0.5 ml). There should
be very little phosphate present (< 10 mM) since it precipitates
in alcohol. Slowly add an equal volume of cold, 70% alcohol
while vortexing. The vortexing and lower final concentration of
alcohol will decrease the clumping problem.
Acetone is used to fix frozen sections for immunoperoxidase.
This is a more gentle fixation than alcohol. You might try
this. Both alcohol and acetone will greatly affect the scatter
from your cells so do not expect them to fall in the same areas
of your cytograms. Also observe the preparation under a
microscope before putting it on the flow to determine whether
the cells are intact, how much debris is present, and whether
they are still fluorescent.
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
