3. 7H10 agar medium in quadrant plates: 7H10 agar medium,
autoclaved, then supplemented with 10% v/v OADC and
100 μg/mL cycloheximide (see Notes 3 and 4). Using a
pipette, dispense ~5 mL of medium into each quadrant of
100 mm quadrant petri dishes, cover and stack plates and
allow the plates to dry at least 48 h (see Note 4).
4. 96-Well tissue culture treated assay plate.
5. P1000 (1 mL) pipettor and pipette tips.
6. Ziploc
® bags.
2.11 Imaging M.
tuberculosis-Infected
Macrophages
1. Round German glass coverslips, 12 mm diameter, #1 thickness
(Chemglass Inc., Vineland, NJ).
2. Premium grade, frosted microscope slides 3
00
 1
00
 1 mm.
3. ProLong
® Gold antifade reagent (Life Technologies, Grand
Island, NY).
4. Tissue culture-grade PBS pH 7.4 without calcium chloride or
magnesium chloride.
5. 4% paraformaldehyde (PFA) in PBS (see Note 5): Reagentgrade, crystalline paraformaldehyde (Sigma-Aldrich Corp.,
St. Louis, MO). Tissue culture-grade PBS, pH 7.4.
6. 24-Well tissue culture-treated assay plates.
7. 23g1 syringe needle (see Note 6).
8. Forceps.
9. Clear, fast drying nail polish.
10. Ziploc
® bags.
3 Methods
3.1 Isolation of Bone
Marrow from Mice
1. Euthanize the mouse using an approved method, such as carbon dioxide asphyxiation followed by a secondary method of
euthanasia, such as cervical dislocation (see Note 7).
2. Isolate the ilium, femur, and tibia bones and clean the muscle
from the bones. Separate the femur from the socket of the ilium
and cut the tibia from the femur at the knee with the small
forceps and scissors. Take care to not break any of the bones.
3. Move the bones to a new petri dish filled with DMEM and
remove any remaining tissue from the bones.
4. From a 50 mL conical tube, fill a 10 mL Luer lock syringe with
fresh DMEM and attach a 23 G Â 1
00 (23g1) needle.
5. Using the small forceps and scissors, cut open both ends of the
bone, insert the needle into one end and flush the bone marrow
into a clean 50 mL conical tube.
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Benjamin K. Johnson et al.
autoclaved, then supplemented with 10% v/v OADC and
100 μg/mL cycloheximide (see Notes 3 and 4). Using a
pipette, dispense ~5 mL of medium into each quadrant of
100 mm quadrant petri dishes, cover and stack plates and
allow the plates to dry at least 48 h (see Note 4).
4. 96-Well tissue culture treated assay plate.
5. P1000 (1 mL) pipettor and pipette tips.
6. Ziploc
® bags.
2.11 Imaging M.
tuberculosis-Infected
Macrophages
1. Round German glass coverslips, 12 mm diameter, #1 thickness
(Chemglass Inc., Vineland, NJ).
2. Premium grade, frosted microscope slides 3
00
 1
00
 1 mm.
3. ProLong
® Gold antifade reagent (Life Technologies, Grand
Island, NY).
4. Tissue culture-grade PBS pH 7.4 without calcium chloride or
magnesium chloride.
5. 4% paraformaldehyde (PFA) in PBS (see Note 5): Reagentgrade, crystalline paraformaldehyde (Sigma-Aldrich Corp.,
St. Louis, MO). Tissue culture-grade PBS, pH 7.4.
6. 24-Well tissue culture-treated assay plates.
7. 23g1 syringe needle (see Note 6).
8. Forceps.
9. Clear, fast drying nail polish.
10. Ziploc
® bags.
3 Methods
3.1 Isolation of Bone
Marrow from Mice
1. Euthanize the mouse using an approved method, such as carbon dioxide asphyxiation followed by a secondary method of
euthanasia, such as cervical dislocation (see Note 7).
2. Isolate the ilium, femur, and tibia bones and clean the muscle
from the bones. Separate the femur from the socket of the ilium
and cut the tibia from the femur at the knee with the small
forceps and scissors. Take care to not break any of the bones.
3. Move the bones to a new petri dish filled with DMEM and
remove any remaining tissue from the bones.
4. From a 50 mL conical tube, fill a 10 mL Luer lock syringe with
fresh DMEM and attach a 23 G Â 1
00 (23g1) needle.
5. Using the small forceps and scissors, cut open both ends of the
bone, insert the needle into one end and flush the bone marrow
into a clean 50 mL conical tube.
172
Benjamin K. Johnson et al.