4. To differentiate myeloid progenitor cells into terminally differentiated macrophages, estradiol must be washed from the cells.
To do this, collect cells in sterile 15 mL conical tube and
centrifuge at 500 Â g for 5 min.
5. Discard supernatant and respend in 5 mL sterile PBS. Centrifuge cells again and discard supernatant. Wash with PBS 3Â
total.
6. After final wash, resuspend in 10 mL macrophage medium and
count cells on hemactyometer.
7. Plate five million cells per plate in a 100 mm non-TC-treated
petri dish and bring up to 10 mL total volume per plate with
macrophage medium. Fresh media should be added 3–4 days
post-differentiation.
8. HoxB8 cells are fully differentiated after 7–10 days in macrophage medium. For subsequent experiments, differentiated
macrophages should be plated in tissue culture-treated petri
dishes.
9. To lift cells from non-TC-treated petri dish, aspirate macrophage media and gently wash with 4 mL PBS.
10. Add 5 mL ice-cold PBS–EDTA to each plate and incubate at
room temperature 5–10 min, or until cells can be easily lifted
by vigorous pipetting.
11. Collect lifted cells and centrifuge at 500 Â g for 5 min and
resuspend in an appropriate volume D10.
12. Cells can be counted and plated in TC-treated dishes with D10
for any further experiments.
13. If the cells are to be lifted from the TC-treated dish,
pre-warmed Accutase (Sigma-Aldrich) should be used to effectively lift the cells while maintaining high viability.
3.6 Isolation,
Maintainance, and
Passage of Max Planck
Institute (MPI) Alveolar
Macrophage-Like Cells
1. MPI cells are generated from the fetal liver cells from mice.
Timed breedings should first be set up following animal guidelines and protocols for each institution.
2. After 14–15 gestational days, euthanize the mother mouse
with a minimum of 10 min CO 2 exposure to ensure death of
both the mother and neonatal mice (see Note 7).
3. Use scissors to cut through the mother’s skin and identify the
murine fetuses.
4. Remove the outer sacs and using surgical tools, carefully extract
the liver, which is easily identified as the large, dark-red organ
present in each fetus. Be sure to remove as much non-liver
tissue as possible from the liver.
5. Transfer whole liver to Eppendorf tube containing 1.0 mL
ice-cold PBS.
Macrophage Infection Models for Mycobacterium tuberculosis
175
To do this, collect cells in sterile 15 mL conical tube and
centrifuge at 500 Â g for 5 min.
5. Discard supernatant and respend in 5 mL sterile PBS. Centrifuge cells again and discard supernatant. Wash with PBS 3Â
total.
6. After final wash, resuspend in 10 mL macrophage medium and
count cells on hemactyometer.
7. Plate five million cells per plate in a 100 mm non-TC-treated
petri dish and bring up to 10 mL total volume per plate with
macrophage medium. Fresh media should be added 3–4 days
post-differentiation.
8. HoxB8 cells are fully differentiated after 7–10 days in macrophage medium. For subsequent experiments, differentiated
macrophages should be plated in tissue culture-treated petri
dishes.
9. To lift cells from non-TC-treated petri dish, aspirate macrophage media and gently wash with 4 mL PBS.
10. Add 5 mL ice-cold PBS–EDTA to each plate and incubate at
room temperature 5–10 min, or until cells can be easily lifted
by vigorous pipetting.
11. Collect lifted cells and centrifuge at 500 Â g for 5 min and
resuspend in an appropriate volume D10.
12. Cells can be counted and plated in TC-treated dishes with D10
for any further experiments.
13. If the cells are to be lifted from the TC-treated dish,
pre-warmed Accutase (Sigma-Aldrich) should be used to effectively lift the cells while maintaining high viability.
3.6 Isolation,
Maintainance, and
Passage of Max Planck
Institute (MPI) Alveolar
Macrophage-Like Cells
1. MPI cells are generated from the fetal liver cells from mice.
Timed breedings should first be set up following animal guidelines and protocols for each institution.
2. After 14–15 gestational days, euthanize the mother mouse
with a minimum of 10 min CO 2 exposure to ensure death of
both the mother and neonatal mice (see Note 7).
3. Use scissors to cut through the mother’s skin and identify the
murine fetuses.
4. Remove the outer sacs and using surgical tools, carefully extract
the liver, which is easily identified as the large, dark-red organ
present in each fetus. Be sure to remove as much non-liver
tissue as possible from the liver.
5. Transfer whole liver to Eppendorf tube containing 1.0 mL
ice-cold PBS.
Macrophage Infection Models for Mycobacterium tuberculosis
175