Rate and Extent of Helicobacter pylori Phagocytosis - Bacterial Pathogenesis: Methods and Protocols - page 140

apparent within 30 s of microbe binding and engulfment is complete within
2-5 min (depending on particle size) (3,4,5) . Indeed, the speed and efficiency
of phagocytosis distinguish this process from pathogen-driven invasion of other
cell types.
We have shown that Helicobacter pylori has the unusual ability to actively
retard its entry into phagocytes (4,6) . Delayed phagocytosis requires bacterial
protein synthesis, and genes within the cag pathogenicity island have been
implicated in this process (4,6,7,8) . Herein, we describe methods used in
our laboratory to measure the rate and extent of H. pylori phagocytosis that
utilize synchronized phagocytosis, differential staining, and immunofluores-
cence microscopy. Advantages of this assay are the fact that very short infection
times can be evaluated, large numbers of samples can be analyzed relatively
quickly, and measurement of uptake is not coupled to intracellular survival or
residence of bacteria in a specific compartment.
2. Materials
2.1. Macrophage Cultivation
1. The method described here is suitable for primary murine bone marrow-derived
macrophages, resident or elicited peritoneal macrophages, or any other adherent
macrophage or macrophage-like cell line such as J774A.1 and RAW264.7.
2. Tissue-culture media: endotoxin-free Dulbecco's modified Eagles medium
(DMEM), minimal essential medium-alpha (MEM) or Hepes-buffered RPMI-
1640 supplemented with 2 mM l-glutamine, and heat-inactivated fetal bovine
serum (FBS, to 10% final concentration) ( see Note 1 ). Store at 4 °C.
2.2. Growth of Helicobacter pylori
1. Blood agar plates: Adjust trypticase soy agar base to pH 6.0 and then autoclave.
Cool sterilized agar in a 50 °C water bath. Add defibrinated sheep blood to 5%
final concentration immediately prior to pouring plates. Store at 4 °C.
2. Purchase Helicobacter pylori from the American Type Culture Collection
(Manassas, VA). Methods described here were optimized using strains NCTC
11637, 60190, and Tx30a (6) . Clinical isolates of H. pylori can also be used.
3. CampyPak bags (Becton-Dickinson, Sparks, MD, USA) or an incubator that can
achieve a microaerophilic (5% O 2 , 10% CO 2 , 85% N 2 ) atmosphere such as an
Heraeus Trigas (Sorvall Instruments, Thermo Scientific, Wlatham, MA, USA)
equipped with an oxygen electrode.
2.3. Microscopy Supplies
1. Round glass coverslips (12 mm diameter, 1.0 μm thick).
2. Straight needle-point stainless steel forceps (from a surgical supply company).