2.5 Maintenance,
Passage, and
Differentiation of
Conditionally
Immortalized
Estradiol-Inducible
HoxB8 Myeloid
Progenitor Cells
1. D10 medium (500 mL): 450 mL DMEM/high glucose,
+4 mM L-Glutamine, 4500 mg/L L-Glucose, with sodium
pyruvate, 0.1 μM sterile-filtered (Hyclone Laboratories,
Logan, UT), 50 mL premium-grade fetal bovine serum, heat
inactivated, 0.22 μm sterile-filtered.
2. Hox Medium (500 mL): 450 mL D10 medium (Subheading
2.5, item 1), 50 mL sterile-filtered supernatant containing
GM-CSF produced by B16 melanoma cell line (or 40 ng/mL
recombinant GM-CSF) with 2 μM β-estradiol.
3. Macrophage differentiation medium (500 mL): 400 mL D10
medium (Subheading 2.5, item 1), 100 mL sterile-filtered
L929 M-CSF conditioned medium.
4. Cell lifting solution (500 mL): Tissue culture-grade Dulbecco’s PBS pH 7.4 without calcium chloride or magnesium
chloride, 10 mM Ultra Pure EDTA, pH 8.0.
5. 100 mm tissue culture dishes.
6. 100 mm non-treated petri dishes.
7. 0.1 mm deep Brightline hemacytometer (Hausser Scientific,
Horsham, PA).
2.6 Isolation,
Maintainance, and
Passage of Max Planck
Institute (MPI)
Macrophage Cells
1. Tissue culture-grade PBS pH 7.4 without calcium chloride or
magnesium chloride.
2. Surgical tools.
3. 40 μM cell strainer.
4. MPI cell medium (500 mL): RPMI-1640, +2.05 mM L-Glutamine (400 mL) (Hyclone Laboratories, Logan, UT), 50 mL
premium-grade fetal bovine serum, heat inactivated, 0.22 μm
sterile-filtered, 50 mL sterile-filtered B16 GM-CSF
conditioned medium (or 30 ng/mL recombinant GM-CSF),
5 mL 100Â penicillin/streptomycin solution, sterile-filtered.
5. 100 mm tissue culture dishes.
6. 1.5 mL sterile Eppendorf tubes.
7. 50 mL sterile conical tubes.
8. Cell lifting solution (500 mL): (see Subheading 2.5, item 4).
2.7 Activation of
Primary Bone MarrowDerived Macrophages
with IFN-γ and LPS
1. Recombinant murine interferon-gamma (IFN-γ, PeproTech,
Rocky Hill, NJ).
2. Lipopolysaccharide
(LPS,
Sigma-Aldrich
Corp.,
St. Louis, MO).
2.8 Infection of
Primary Bone MarrowDerived Macrophages
with M. tuberculosis in
24-Well Plates
1. OADC (oleic acid, albumin, dextrose, and catalase) supplement (see Note 3).
2. Tween-80 (polysorbate 80).
170
Benjamin K. Johnson et al.
Passage, and
Differentiation of
Conditionally
Immortalized
Estradiol-Inducible
HoxB8 Myeloid
Progenitor Cells
1. D10 medium (500 mL): 450 mL DMEM/high glucose,
+4 mM L-Glutamine, 4500 mg/L L-Glucose, with sodium
pyruvate, 0.1 μM sterile-filtered (Hyclone Laboratories,
Logan, UT), 50 mL premium-grade fetal bovine serum, heat
inactivated, 0.22 μm sterile-filtered.
2. Hox Medium (500 mL): 450 mL D10 medium (Subheading
2.5, item 1), 50 mL sterile-filtered supernatant containing
GM-CSF produced by B16 melanoma cell line (or 40 ng/mL
recombinant GM-CSF) with 2 μM β-estradiol.
3. Macrophage differentiation medium (500 mL): 400 mL D10
medium (Subheading 2.5, item 1), 100 mL sterile-filtered
L929 M-CSF conditioned medium.
4. Cell lifting solution (500 mL): Tissue culture-grade Dulbecco’s PBS pH 7.4 without calcium chloride or magnesium
chloride, 10 mM Ultra Pure EDTA, pH 8.0.
5. 100 mm tissue culture dishes.
6. 100 mm non-treated petri dishes.
7. 0.1 mm deep Brightline hemacytometer (Hausser Scientific,
Horsham, PA).
2.6 Isolation,
Maintainance, and
Passage of Max Planck
Institute (MPI)
Macrophage Cells
1. Tissue culture-grade PBS pH 7.4 without calcium chloride or
magnesium chloride.
2. Surgical tools.
3. 40 μM cell strainer.
4. MPI cell medium (500 mL): RPMI-1640, +2.05 mM L-Glutamine (400 mL) (Hyclone Laboratories, Logan, UT), 50 mL
premium-grade fetal bovine serum, heat inactivated, 0.22 μm
sterile-filtered, 50 mL sterile-filtered B16 GM-CSF
conditioned medium (or 30 ng/mL recombinant GM-CSF),
5 mL 100Â penicillin/streptomycin solution, sterile-filtered.
5. 100 mm tissue culture dishes.
6. 1.5 mL sterile Eppendorf tubes.
7. 50 mL sterile conical tubes.
8. Cell lifting solution (500 mL): (see Subheading 2.5, item 4).
2.7 Activation of
Primary Bone MarrowDerived Macrophages
with IFN-γ and LPS
1. Recombinant murine interferon-gamma (IFN-γ, PeproTech,
Rocky Hill, NJ).
2. Lipopolysaccharide
(LPS,
Sigma-Aldrich
Corp.,
St. Louis, MO).
2.8 Infection of
Primary Bone MarrowDerived Macrophages
with M. tuberculosis in
24-Well Plates
1. OADC (oleic acid, albumin, dextrose, and catalase) supplement (see Note 3).
2. Tween-80 (polysorbate 80).
170
Benjamin K. Johnson et al.