6. Pipet up and down to disrupt liver and transfer cell suspension
to 4.0 mL ice-cold PBS.
7. Filter into 50 mL conical tube through 40 μm cell strainer.
8. Centrifuge at 500 Â g for 5 min, discard supernatant, repeat
PBS wash 2Â.
9. Resuspend in 3.5 mL complete MPI cell media.
10. Plate 2.0, 1.0, and 0.5 mL into six-well plates.
11. Bring volume up to 3.0 mL total with MPI cell media.
12. After 3 days, change media, discard floating cells.
13. After an additional 3 days, monolayer should develop.
14. Detach with 1.5 mM EDTA–PBS and plate onto 100 mm
tissue culture plates.
15. Round cells are the MPI cells. With each passage, more of the
cells in the plates will have the “fried egg” morphology that are
the MPI cells.
3.7 Activation of
Primary Bone MarrowDerived Macrophages
with IFN-γ and LPS
1. Macrophages are activated by IFN-γ and LPS in a two-step
procedure.
2. After scraping macrophages from confluent petri dishes (e.g.,
Subheading 3.2), seed cells into the desired type of assay plate
(see Note 11) and allow to adhere overnight before the addition of activation reagents.
3. Once macrophages are attached to the assay plate, add IFN-γ
to a final concentration of 100 U/mL.
4. Incubate overnight in a humidified incubator at 37
C and 5%
CO 2 .
5. Add LPS at a final concentration of 10 ng/mL and incubate
overnight in a humidified incubator at 37
C and 5% CO 2 .
6. Once activated, the culture medium will turn yellow and the
cells will have an altered morphology.
3.8 Infection of
Primary Bone MarrowDerived Macrophages
with M. tuberculosis in
24-Well Plates
1. Culture M. tuberculosis to mid- to late-log phase (OD 600
0.5–1.0) in Middlebrook 7H9 supplemented with 10% v/v
OADC and 0.05% v/v Tween-80.
2. An OD 600 of 0.6 represents approximately 1 Â 10
8 bacteria
per/mL. Add an appropriate volume of bacteria to obtain
~1 Â 10
8 bacteria in 5 mL of pre-warmed uptake buffer.
3. Centrifuge for 10 min at 1800 Â g.
4. Discard the supernatant and resuspend in 1 mL of pre-warmed
basal uptake buffer in a 15 mL conical tube.
5. Place the tube in a 15 mL conical tube rack to provide support
and carefully syringe the bacterial solution eight times with a
tuberculin syringe (see Note 13). This procedure will unclump
the bacteria into single cells.
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to 4.0 mL ice-cold PBS.
7. Filter into 50 mL conical tube through 40 μm cell strainer.
8. Centrifuge at 500 Â g for 5 min, discard supernatant, repeat
PBS wash 2Â.
9. Resuspend in 3.5 mL complete MPI cell media.
10. Plate 2.0, 1.0, and 0.5 mL into six-well plates.
11. Bring volume up to 3.0 mL total with MPI cell media.
12. After 3 days, change media, discard floating cells.
13. After an additional 3 days, monolayer should develop.
14. Detach with 1.5 mM EDTA–PBS and plate onto 100 mm
tissue culture plates.
15. Round cells are the MPI cells. With each passage, more of the
cells in the plates will have the “fried egg” morphology that are
the MPI cells.
3.7 Activation of
Primary Bone MarrowDerived Macrophages
with IFN-γ and LPS
1. Macrophages are activated by IFN-γ and LPS in a two-step
procedure.
2. After scraping macrophages from confluent petri dishes (e.g.,
Subheading 3.2), seed cells into the desired type of assay plate
(see Note 11) and allow to adhere overnight before the addition of activation reagents.
3. Once macrophages are attached to the assay plate, add IFN-γ
to a final concentration of 100 U/mL.
4. Incubate overnight in a humidified incubator at 37
C and 5%
CO 2 .
5. Add LPS at a final concentration of 10 ng/mL and incubate
overnight in a humidified incubator at 37
C and 5% CO 2 .
6. Once activated, the culture medium will turn yellow and the
cells will have an altered morphology.
3.8 Infection of
Primary Bone MarrowDerived Macrophages
with M. tuberculosis in
24-Well Plates
1. Culture M. tuberculosis to mid- to late-log phase (OD 600
0.5–1.0) in Middlebrook 7H9 supplemented with 10% v/v
OADC and 0.05% v/v Tween-80.
2. An OD 600 of 0.6 represents approximately 1 Â 10
8 bacteria
per/mL. Add an appropriate volume of bacteria to obtain
~1 Â 10
8 bacteria in 5 mL of pre-warmed uptake buffer.
3. Centrifuge for 10 min at 1800 Â g.
4. Discard the supernatant and resuspend in 1 mL of pre-warmed
basal uptake buffer in a 15 mL conical tube.
5. Place the tube in a 15 mL conical tube rack to provide support
and carefully syringe the bacterial solution eight times with a
tuberculin syringe (see Note 13). This procedure will unclump
the bacteria into single cells.
176
Benjamin K. Johnson et al.