Biology Reference
In-Depth Information
and CaCl
2
to remove all non-adherent cells. Wash once with ice-cold D-PBS
without MgCl
2
and CaCl
2
but supplemented with 1 g/L d-glucose. Add 15 mL of
PBS-glucose and incubate the dish on ice with gentle rocking for 10 min. Make
sure the adherent macrophages remain submerged at all time.
7. To retrieve adherent BMMs, pipette the PBS across the entire surface of the plate
and monitor under the microscope that all BMMs have been detached. Avoid
bubbles as much as possible. Transfer the BMM suspension into a 50-mL conical
tube and centrifuge the cells at 200
×g
for 5 min at 15 °C. Discard the supernatant
and gently resuspend the cell pellet in 5 mL of DMEM.
8. Count BMMs using a phase hemacytometer and seed 12-mm glass coverslips in
wells of a 24-well plate at a density of 1 × 10
5
/well in complete medium. You
should obtain 1-2 × 10
7
BMMs/dish.
9. Replenish medium after 24 h in culture. The cells may then undergo one or
two rounds of multiplication and then stop multiplying. On day 7 post-isolation,
BMMs are ready to use (
see
Note 4
).
3.2. Macrophage Infections
1. The following method of macrophage infection with either
B. abortus
or
F. tularensis
aims at synchronizing bacterial phagocytosis, which is important
for subsequent intracellular trafficking analyses, as all bacteria have entered
macrophages within a short time frame.
2. To prepare bacterial inocula, streak
B. abortus
first on TSA plates from a frozen
stock vial and grow for 3 days at 37 °C. Then inoculate 2 mL of TSB in a 50-mL
conical tube with two to three fresh isolated colonies and incubate for 16 h in a
shaking incubator at 37 °C. Grow
F. tularensis
on CHAB plates from a frozen
stock vial for 3 days at 37 °C in 7% CO
2
. Right before proceeding for infections,
resuspend a few colonies in TSB containing 0.1% l-cysteine. Estimate bacterial
counts by measuring the optical density at 600 nm (OD
600 nm
)ofthe
Brucella
sub-culture or the
Francisella
suspension. An OD
600 nm
of 1.0 corresponds to
∼
3×10
9
bacteria/mL for both organisms, but this needs to be determined for
every organism by plating serial dilutions of the bacterial inoculum on appropriate
medium before hand. Determining the bacterial inoculum is essential to calculate
the multiplicity of infection (MOI) to be used. Theoretical MOIs of 25 and 50
are used for
B. abortus
and
F. tularensis
, respectively (
see
Note 5
).
3. To infect BMMs, pre-chill macrophages for 5 min on ice while preparing
the bacterial suspension in ice-cold complete medium. On ice, aspirate the
macrophage medium in one well at a time and immediately replace with 0.5 mL
of bacterial suspension. Centrifuge bacteria onto BMMs at 400
×g
for 10 min at
4 °C to favor bacteria-macrophage contact.
4. Rapidly warm BMMs to 37 °C for 3 min by floating the plates in a waterbath
to trigger bacterial phagocytosis. Transfer plates to a tissue-culture incubator and
further incubate them for a total of 20 min at 37 °C under 7% CO
2
.(
see
Note 6
).
5. Wash BMMs five times with DMEM to remove extracellular bacteria, incubate
for 40 min in complete medium and then for 60 min in complete medium
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