6. Spin down the bone marrow in a swinging bucket, refrigerated
centrifuge, at 200 Â g for 10 min at 4
C. Remove supernatent
and gently resuspend bone marrow in 8 mL of bone marrow
macrophage medium pre-warmed to 37
C.
7. Allow larger particles to settle for approximately 1–2 min.
8. Add 1 mL of resuspended bone marrow to 9 mL bone marrow
macrophage medium in a 100 mm petri dish and swirl dish to
disperse the cells.
9. Incubate in a humidified incubator at 37
C and 5% CO 2 .
10. Add 5 mL of fresh bone marrow macrophage medium,
pre-warmed to 37
C, to each dish after 3–4 days of incubation.
Monitor the presence of adherent cells using an inverted
microscope. The macrophage monolayers will be confluent
after 5–7 days.
3.2 Expansion and
Passaging of Primary
Bone Marrow-Derived
Macrophages
1. Once the cells are confluent, remove the medium and discard.
2. Add 10 mL of tissue culture-grade PBS, prechilled to 4
C, to
each dish.
3. Incubate at 4
C for 10 min (see Note 8).
4. Remove the adherent cells using a cell scraper (see Note 9).
5. Collect the cell suspensions into a 50 mL conical tube.
6. Spin down the cell suspension in a swinging bucket, refrigerated centrifuge, pre-chilled to 4
C, at 200 Â g for 10 min.
7. Discard supernatant and gently resuspend cell pellet in fresh,
pre-warmed bone marrow macrophage medium.
8. Remove 10 μL and load onto a hemacytometer for counting
the number of cells. A typical confluent 100 mm petri dish will
have approximately 1–1.5 Â 10
6 cells/mL (see Note 10).
9. Cells can be split up to 1:8.
10. For seeding 24-, 96-, and 384-well assay plates, see Note 11 for
appropriate cell concentrations.
3.3 Maintenance and
Passage of J774 Cells
1. Thaw a vial of frozen J774 cells in a 37
C water bath. Add the
entire volume of the vial to 10 mL of pre-warmed D10 medium
in a 100 mm petri dish.
2. Incubate overnight in a humidified incubator at 37
C and 5%
CO 2 .
3. Remove the medium and replace with 10 mL of fresh D10
medium (to remove any residual DMSO from the freezing
medium) and incubate at 37
C and 5% CO 2 until the cells
become confluent.
4. Passage cells as described above (see Subheading 3.2) for the
primary murine bone marrow-derived macrophages, with the
Macrophage Infection Models for Mycobacterium tuberculosis
173
centrifuge, at 200 Â g for 10 min at 4
C. Remove supernatent
and gently resuspend bone marrow in 8 mL of bone marrow
macrophage medium pre-warmed to 37
C.
7. Allow larger particles to settle for approximately 1–2 min.
8. Add 1 mL of resuspended bone marrow to 9 mL bone marrow
macrophage medium in a 100 mm petri dish and swirl dish to
disperse the cells.
9. Incubate in a humidified incubator at 37
C and 5% CO 2 .
10. Add 5 mL of fresh bone marrow macrophage medium,
pre-warmed to 37
C, to each dish after 3–4 days of incubation.
Monitor the presence of adherent cells using an inverted
microscope. The macrophage monolayers will be confluent
after 5–7 days.
3.2 Expansion and
Passaging of Primary
Bone Marrow-Derived
Macrophages
1. Once the cells are confluent, remove the medium and discard.
2. Add 10 mL of tissue culture-grade PBS, prechilled to 4
C, to
each dish.
3. Incubate at 4
C for 10 min (see Note 8).
4. Remove the adherent cells using a cell scraper (see Note 9).
5. Collect the cell suspensions into a 50 mL conical tube.
6. Spin down the cell suspension in a swinging bucket, refrigerated centrifuge, pre-chilled to 4
C, at 200 Â g for 10 min.
7. Discard supernatant and gently resuspend cell pellet in fresh,
pre-warmed bone marrow macrophage medium.
8. Remove 10 μL and load onto a hemacytometer for counting
the number of cells. A typical confluent 100 mm petri dish will
have approximately 1–1.5 Â 10
6 cells/mL (see Note 10).
9. Cells can be split up to 1:8.
10. For seeding 24-, 96-, and 384-well assay plates, see Note 11 for
appropriate cell concentrations.
3.3 Maintenance and
Passage of J774 Cells
1. Thaw a vial of frozen J774 cells in a 37
C water bath. Add the
entire volume of the vial to 10 mL of pre-warmed D10 medium
in a 100 mm petri dish.
2. Incubate overnight in a humidified incubator at 37
C and 5%
CO 2 .
3. Remove the medium and replace with 10 mL of fresh D10
medium (to remove any residual DMSO from the freezing
medium) and incubate at 37
C and 5% CO 2 until the cells
become confluent.
4. Passage cells as described above (see Subheading 3.2) for the
primary murine bone marrow-derived macrophages, with the
Macrophage Infection Models for Mycobacterium tuberculosis
173