Rate and Extent of Helicobacter pylori Phagocytosis - Bacterial Pathogenesis: Methods and Protocols - page 142

3.2. Plating Macrophages on Coverslips
Helicobacter pylori will infect different types of macrophages including
primary human and murine macrophages and murine macrophage-like cell lines
and human neutrophils isolated from peripheral blood (4,6,8,9,10,11,12,13) .
As noted above, endotoxin-free reagents are required to prevent unintended
cell activation. In addition, tissue-culture medium must not contain penicillin
or streptomycin, as these drugs compromise H. pylori viability. Thus, all
phagocytes should be maintained in antibiotic-free medium or, at a minimum,
transferred to antibiotic-free medium 48 h prior to infection.

1. Flaming of coverslips and dispersal in 35-mm dishes. One 35-mm tissue-
culture dish is needed per sample. Each dish will hold three coverslips that
provide triplicate samples for each experimental condition or time point. Using
forceps, remove a few coverslips from their ethanol storage bottle and place
onto a pile of Kimwipes. Fill one small microbeaker with 70% ethanol and
light a Bunsen burner. Working with one coverslip at a time, grasp with
forceps, dunk into ethanol, and drain off excess ethanol by touching edge of
the coverslip to the pile of Kimwipes. Pass the coverslip through the flame
and then place it into a 35-mm dish. Because the 35-mm dishes are opened
only briefly, this procedure can be performed on the bench top. Failure to drain
off excess ethanol will cause coverslips to shatter when passed through the
flame.
2. Plating macrophages: Harvest BMM, J774A.1, or RAW264.7 cells using a cell
scraper. Harvest peritoneal macrophages from anaesthetized mice by lavage with
4 °C PBS. Centrifuge all cells at 400 × g for 15 min at 4 °C. Resuspend the cell
pellets in tissue-culture medium (DMEM for J774A.1 and RAW264.7, MEM
for peritoneal macrophages, DMEM or MEM supplemented with CSF-1 for
bone marrow-derived macrophages, and RPMI-1640 for human macrophages) to
achieve

5-7×10 5 cells/mL. A moderate cell density is optimal for microscopy.
Working in the tissue-culture hood, remove lids from the 35-mm dishes. Be sure
that coverslips in each dish are not touching one another or the edge of the
dish. If necessary, reposition coverslips using a sterile probe (a yellow Pipetman
tip or forceps can be used to push coverslips into position). Plate 50 μL of
the macrophage suspension directly onto each coverslip ( see Note 4 ). Liquid
should spread evenly over the acid-washed glass, and surface tension will hold
the domes of liquid in place ( see Note 5 ). Close dishes and transfer into a
37 °C tissue-culture incubator for 90-120 min to let cells attach. Dishes can
be placed in a small pan or on a tray to facilitate transport. Thereafter, add
1.5-2 mL of additional medium to each dish and leave in the tissue-culture
incubator overnight. For peritoneal macrophages, remove non-adherent lympho-
cytes using three changes of medium or PBS before addition of 1.5-2 mL of fresh
medium.

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