Rate and Extent of Helicobacter pylori Phagocytosis - Bacterial Pathogenesis: Methods and Protocols - page 144

incubator. Dilute H. pylori in cold tissue-culture medium to achieve a moderate
multiplicity of infection (MOI). From an MOI of 15:1, we obtain 8 bacteria

per cell. Add 1 mL of cold, diluted H. pylori to each dish of macrophages
(already containing 1 mL of warm medium). Mixing warm and cold medium
reduces the overall temperature below the threshold for phagocytosis (16 °C).
Rapidly and carefully transfer the dishes onto microplate carriers in a refrig-
erated tissue-culture centrifuge pre-cooled to 10-12 °C. Centrifuge for 3-4 min
at 600 × g with maximum braking. Carefully transfer dishes to 37 °C incubator
to allow phagocytosis or transfer immediately to ice ( t = 0 min sample). If
dishes remain cold during centrifugation, bacteria in the 0-min samples should
be cell associated but not internalized.

∼

3.5. Differential Staining of Bound and Ingested Bacteria
The method described here stains sequentially extracellular and intracellular
bacteria and as such allows the rate and extent of phagocytosis to be quantified.
1. At the desired time points (e.g., 0, 1, 3, 5, 7, 10, 20, 30 min; determine empirically
for each strain and cell type) move samples from 37 °C into a metal pan placed
over a bed of ice. Working quickly, aspirate medium and wash samples with three
changes of 4 °C PBS to remove unbound organisms and stop further uptake of
any uningested, surface-associated bacteria.
2. Dilute anti- H. pylori Ab in PBS/glucose: We use pAb YVS6601 at 1:2000 for
strains 11637 and 60190 and 1:750 for strain Tx30a. Aspirate cold PBS from each
dish and replace with 1 mL of antibody solution. Leave dishes on a bed of ice
and incubate in a refrigerator or cold room for 30-60 min ( see Note 7 ). Because
the macrophages are intact, the Ab has access only to extracellular organisms.
3. Aspirate the primary Ab solution from each dish and wash samples gently
with three changes of ice-cold PBS/glucose. Fix and permeabilize samples using
methanol: Pour 3 mL of methanol into each dish and let sit for 10 min. From

this point on, samples can be manipulated at room temperature. Aspirate off the
methanol, rinse samples once with PBS to rehydrate and then block in PAB for
15-30 min.
4. While samples are blocking, dilute the rhodamine-conjugated secondary Ab 1:200
in PAB. Thirty microliters is needed per coverslip. Also, set up a pile of Kimwipes
and label a 24-well dish lid that will be used to hold coverslips during remaining
incubations ( see Fig. 1 ).
5. Working with one dish at a time, pick up a coverslip using forceps and drain
off PAB by touching the edge of the coverslip to the pile of Kimwipes. Dry
the back of each coverslip and place cell-side up on the 24-well dish lid ( see
Fig. 1 ). Overlay with 30 μL of secondary Ab. Repeat for the other samples. Place
coverslips inside a Tupperware type box lined with damp paper towels to prevent
evaporation of liquid. Incubate for 30-60 min at room temperature. At the end of
this incubation, extracellular H. pylori will be stained red. Meanwhile, prepare a

∼