PMN markers

Sun Nov 3 15:55:20 EST 1996

A convenient and simple way to identify PMN cells is based on
analysis of the lysosomal proton pump. Granules of PMN cells
(including baso- and acidophils) show similar properties as lysosomes
in terms of active accumulation of the lysosomotrophic cationic
probes. Such a probe is acridine orange (AO). The uncharged AO
molecules enter live cells but after their protonation (which happens
at low pH of the lysosomes) they become entrapped therein. Incubation
of live cells (cells have to be live, not fixed or permeabilized!)
with 1 ug/ml of AO results in selective accumulation of AO in
lysosomes which fluoresce red. All other structures (RNA, DNA)
fluoresce green at such low AO concentration. PMNs show several fold
higher red (>640 nm) fluorescence compared to monocytes, which in
turn have significantly higher fluorescence than lymphocytes after 5
- 10 min incubation with AO. The method is described in Vol 41 Meth
Cell Biol., Chapter 12, pp 185-195.
Zbigniew Darzynkiewicz

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