Biology Reference
In-Depth Information
whole blood from the same donor and 2 mL of RosetteSep Monocyte Enrichment
cocktail (
see
Note 13
). Incubate on a tube rotator for 20 min at room temperature.
3. Dilute mixture 1:2 with PBS containing 2% FBS and carefully layer 25 mL onto
25 mL of Ficoll-Paque Plus in a 50-mL conical tube. Centrifuge for 20 min at
1200 ×
g
with the brake off. After centrifugation, a layer of highly enriched
monocytes will be found at the Ficoll-PBS interface. Carefully remove this layer
with a pipette and transfer to a new 50-mL conical tube. Wash monocytes twice
with PBS containing 2% FBS, centrifuging at 500 ×
g
in between each wash
step. Resuspend cells in RPMI plus 10% FBS and 50 ng/mL M-CSF to a final
concentration of 0.5-1.0 × 10
6
cells/mL (should require approximately 40 mL of
media). Transfer to a T-150 tissue culture flask and incubate at 37 °C in 5% CO
2
.
Add fresh M-CSF to the flask after 3 days of incubation.
4. Note the monocytes will differentiate into adherent macrophages by day 7 of
incubation. To harvest cells, discard the culture medium and wash once with 25
mL of cold PBS without Ca
++
or Mg
++
. Add 20 mL of fresh cold PBS to the
flask and incubate on ice until cells begin to round-up and detach (usually 15-20
min) (
see
Note 14
). Gently scrape cells into the PBS with a cell scraper and
transfer the cell suspension to a 50-mL conical centrifuge tube. Pellet the cells
by centrifugation at 500 ×
g
for 5 min. Resuspend cells in 5-10 mL of RPMI
plus 10% FBS and 50 ng/mL M-CSF. Count cells, add medium to adjust to the
desired cell concentration, and re-plate in a tissue culture plate (
see
Note 15
).
Incubate at 37 °C in 5% CO
2
for at least6htoallow macrophages to adhere.
3.4. Infection of Monocyte-Derived Macrophages with Coxiella
burnetii
1. A good infection of macrophages can be achieved by direct addition of purified
C. burnetii
to the culture medium. To achieve near 100% infection, multiplicities
of infection of approximately 10 and 100 are required for avirulent phase II and
virulent phase I strains, respectively (
see
Note 1
). Add the appropriate amount of
bacteria to each well and mix by gently rocking and swirling the plate. Incubate
the plate at 37 °C in 5% CO
2
4-24 h.
2. Gently wash the cells three times with PBS or culture medium to remove extra-
cellular bacteria.
3. Add fresh culture medium and incubate at 37 °C in 5% CO
2
to allow
C. burnetii
replication and vacuole development. Replication vacuoles should be visible
by phase contrast microscopy within 2-3 days post-infection. At 6 days post-
infection, growth of
C. burnetii
should have reached stationary phase and essen-
tially all cells will contain large bacteria-filled vacuoles (
see
Fig. 2
).
3.5. Enumeration of Coxiella burnetii Replication
1. Conduct quantification of
C. burnetii
replication in macrophages by measuring
the number of bacterial genome equivalents by quantitative PCR. Total (host
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