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> When labeling leukocyte cell surface antigens with the appropriate
> conjugated antibody, is it best to fix (with paraformaldehyde) the cell
> suspension:
> a) before antibody labeling?
> b) after antibody labeling?
I believe the usual procedure would be to suspend the cells in the
fixative after the final wash. If the cells are then left *permanently*
in that fixing solution, the question of *time* of fixation is no longer
important (since staining intensity should not be changed from time 0
onwards).
> c) what concentration of paraformaldehyde (1% final concentration?)
> and is it dependent upon the
> d) cell type?
> e) antigen to which the antibody is specific for?
> f) monoclonal vs. polyclonal antibody?
The fixative used here for the last decade and a half (and dubbed
"FACS-FIX") but for which I cannot now quote a source, is as follows:
10g glucose
13ml formalin (40% formaldehyde solution)
2.5ml 10% sodium azide
Make up to 500ml with PBS.
The glucose keeps the cells puffed up so their light scatter is
preserved. The azide prevents bugs from partying on the glucose. We
use this for all cell types and surface stains. The cells remain
intact for many months, but a "yellowish" (seen in both FITC and PE
detectors) background fluorescence does slowly appear (intensity of
which is less than about 1000 "molecules of equivalent soluble
fluorescein" after 7 days).
I can't advise on fixation of *intra-cellular* stained cells.
Regards,
\ / < Flow Systems Laboratory
Frank Battye \__/ <<<<< The Walter & Eliza Hall Institute
Robyn Muir ----------!!<<<<<<<< Voice: 61_3_9345 2540
Dora Constantinou /!!\ <<<<< Fax: 61_3_9347 0852
o !! \ < IN:: "facs_copy@wehi.edu.au"
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