Rate and Extent of Helicobacter pylori Phagocytosis - Bacterial Pathogenesis: Methods and Protocols - page 148

opsonize with IgG, incubate washed organisms with a sub-agglutinating concen-
tration Ab directed against H. pylori LPS (or another surface antigen) for 30 min
at 37 °C. To opsonize bacteria with complement factors, incubate washed bacteria
in 50% fresh human serum for 20-30 min at 37 °C. Relatively high concen-
trations of serum are required to fix complement on this organism (17) . Wash
all opsonized bacteria prior to measurement of optical density and macrophage
infection.
7. Human monocyte-derived macrophages tend to lift off coverslips when incubated
at 4 °C. Thus, human macrophages (or other cells that do not adhere strongly
such as neutrophils) should be fixed in 2% paraformaldehyde for 15 min at room
temperature and then washed with PBS. Incubation with primary Ab can then be
performed at room temperature. Subsequent steps of the protocol are unchanged.
8. When using pharmacological inhibitors of macrophage signaling pathways, it is
important to note that many of these agents are inactivated by components in
tissue-culture medium and/or FBS. Thus, for these types of studies, it is necessary
to use serum-free medium or Hank's-buffered saline solution supplemented with
1m M Hepes and 10 m M glucose. Washed macrophages should be serum-starved
or incubated in Hank's buffer for at least 2 h prior to inhibitor treatment and
infection. In general, macrophages are pretreated with inhibitor (or vehicle) for
30-60 min at 37 °C and then infected in inhibitor-containing medium. Incubate
samples at 37 °C for 30 min prior to differential staining.
Acknowledgements
This work was supported by a Merit Review Grant from the Department of
Veteran's Affairs.
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