Rate and Extent of Helicobacter pylori Phagocytosis - Bacterial Pathogenesis: Methods and Protocols - page 147

macrophages). It is important to note that “0-min” samples should contain <3%
ingested bacteria ( see Fig. 2 ), and the number of total cell-associated bacteria
should remain constant over the course of the experiment as the fraction of intra-
cellular H. pylori increases. If large numbers of “green-only” bacteria are apparent
at 0 min, either samples did not remain cool (<16 °C) during the centrifugation
or an artifact occurred because the anti- H. pylori antibody used in the first round
of staining did not react efficiently with surface exposed epitopes. These caveats
aside, this assay is rapid and efficient and can be adapted easily to assess the effects
of opsonins or signaling pathway inhibitors on bacterial binding and phagocy-
tosis ( see Note 8 ) to compare phagocytosis of different strains of bacteria or to
compare H. pylori entry into different types of phagocytes ( see Fig. 2 ) (6,14) .
Finally, the fact that this assay is independent of phagosome composition is signif-
icant as some, but not all, strains of H. pylori inhibit phagosome maturation and
because phagosome composition varies somewhat in primary macrophages and
macrophage-like cell lines (10,11) .
4. Notes
1. To prevent macrophage priming or activation, it is essential to ensure that all
tissue-culture media, serum, and other reagents are endotoxin free.
2. The anti- H. pylori Ab we prefer works well with many H. pylori strains and is cost
effective. However, any Ab that reacts with both surface and cytoplamic (internal)
bacterial epitopes would be suitable. Alternatively, two different Ab may be used
to detect intracellular and extracellular organisms. In all cases, each Ab should
be tested for reactivity with intact and methanol-permeabilized bacteria and the
appropriate concentrations determined prior to use with infected macrophages.
3. We prefer the polyvinyl alcohol base of gelvatol mounting medium because
it hardens rapidly and completely. The mounting medium we prepare gives
consistent results, but, in our hands, this is not true for similar products obtained
from commercial vendors. A common alternative to gelvatol is buffered glycerol
(with or without an anti-fading agent). Because glycerol mounting solutions do
not harden, coverslips must be attached to microscope slides using several layers
of nail polish (which is time consuming to apply and apt to leak).
4. To use this assay with phagocytes that do not adhere strongly to uncoated glass,
coverslips should be precoated with extracellular matrix proteins. For example,
plate human monocyte-derived macrophages on coverslips coated with 0.1 mg/mL
collagen and plate human neutrophils on coverslips coated with 0.1 mg/mL
fibrinogen.
5. This plating method conserves macrophages, ensures that each sample contains a
similar number of cells and (most importantly) is compatible with synchronized
phagocytosis.
6. Helicobacter pylori are not opsonized in vivo (15,16) . Nevertheless, bacteria can
be opsonized with specific IgG or complement factors in vitro (6,13,17) .To