AJP - Cell Physiology, Vol 252, Issue 6 C677-C683, Copyright © 1987 by American Physiological Society
Measurement of superoxide release from single pulmonary alveolar macrophages
K. A. DiGregorio, E. V. Cilento and R. C. Lantz
An electrooptical method was developed to quantify superoxide (O2-) release
from single rat pulmonary alveolar macrophages (PAM) during adherence to
the bottom of a culture dish. This was done by measuring the reduction of
nitro blue tetrazolium (NBT) to a diformazan precipitate at 550 nm from
videorecorded images of individual cells. Temporal changes in cell optical
density, which are proportional to the mass of diformazan produced, were
calculated from videophotometric measurements of the change in light
intensity over individual cells. Total diformazan produced increased 78 and
126% with an increase in NBT from 0.5 to 1.0 and 2.0 mg/ml, respectively.
Total diformazan produced and maximum rate of production among individual
PAM varied two- to threefold providing strong evidence for heterogeneity in
O2- production. Specific inhibition of O2- production by superoxide
dismutase, iodoacetate, and chlorpromazine significantly reduced the total
diformazan produced and maximum rate of diformazan production. Hydrogen
peroxide was not involved in NBT reduction, since catalase alone did not
significantly change diformazan production. This novel method to quantify
O2- release from single PAM should be valuable in analyzing heterogeneity
and single cell kinetics of O2- production, in assessing the effects of
exposure of cells to particulates on O2- release, and in relating release
to electrophysiological measurements.
