4. 50 mL sterile conical tubes.
5. 23 gauge  1
00 (23g1) syringe needle.
6. 10 mL Luer lock syringe.
7. Small scissors and forceps.
2.2 Expansion and
Passaging of Primary
Bone Marrow-Derived
Macrophages
1. Bone marrow macrophage medium (500 mL) (see Subheading
2.1, item 1).
2. Tissue culture-grade phosphate-buffered saline (PBS) pH 7.4
without calcium chloride or magnesium chloride.
3. Small (18 mm) blade cell scraper.
4. INCYTO C-Chip disposable hemacytometer.
5. 100 mm petri dishes.
6. 50 mL sterile conical tubes.
7. If seeding 24-, 96-, or 384-well assay plates: 24-well tissue
culture-treated plates; 96- and 384-well black, clear, flatbottom tissue culture-treated assay plates (Corning, Corning,
NY); 96- and 384-well white, flat-bottom, low flange tissue
culture-treated assay plates (Corning, Corning, NY).
2.3 Maintenance and
Passage of J774 Cells
1. D10 medium (500 mL): 435 mL DMEM with 4.5 g/L glucose and without sodium pyruvate and L-glutamine, 5 mL
100 mM sodium pyruvate, 5 mL 200 mM L-glutamine, 5 mL
100Â penicillin (10,000 I.U./mL) streptomycin (10,000 μg/
mL), 50 mL heat inactivated, low-endotoxin FBS. Sterilize by
filtering through 0.22 μm filter.
2. Tissue culture-grade PBS pH 7.4 without calcium chloride or
magnesium chloride.
3. Greiner Bio-One small (18 mm) blade cell scraper.
4. INCYTO C-Chip disposable hemacytometer.
5. 100 mm petri dishes.
6. 50 mL sterile conical tubes.
2.4 Maintenance and
Passage of THP-1 Cells
1. RPMI complete medium (500 mL): 445 mL Roswell Park
Memorial Institute (RPMI) 1640, 5 mL 100Â penicillin
(10,000 I.U./mL) streptomycin (10,000 μg/mL), 50 mL
heat inactivated, low-endotoxin FBS. Sterilize by filtering
through 0.22 μm filter.
2. INCYTO C-Chip disposable hemacytometer.
3. 100 mm petri dishes (or tissue culture flasks).
4. 50 mL sterile conical tubes.
Macrophage Infection Models for Mycobacterium tuberculosis
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5. 23 gauge  1
00 (23g1) syringe needle.
6. 10 mL Luer lock syringe.
7. Small scissors and forceps.
2.2 Expansion and
Passaging of Primary
Bone Marrow-Derived
Macrophages
1. Bone marrow macrophage medium (500 mL) (see Subheading
2.1, item 1).
2. Tissue culture-grade phosphate-buffered saline (PBS) pH 7.4
without calcium chloride or magnesium chloride.
3. Small (18 mm) blade cell scraper.
4. INCYTO C-Chip disposable hemacytometer.
5. 100 mm petri dishes.
6. 50 mL sterile conical tubes.
7. If seeding 24-, 96-, or 384-well assay plates: 24-well tissue
culture-treated plates; 96- and 384-well black, clear, flatbottom tissue culture-treated assay plates (Corning, Corning,
NY); 96- and 384-well white, flat-bottom, low flange tissue
culture-treated assay plates (Corning, Corning, NY).
2.3 Maintenance and
Passage of J774 Cells
1. D10 medium (500 mL): 435 mL DMEM with 4.5 g/L glucose and without sodium pyruvate and L-glutamine, 5 mL
100 mM sodium pyruvate, 5 mL 200 mM L-glutamine, 5 mL
100Â penicillin (10,000 I.U./mL) streptomycin (10,000 μg/
mL), 50 mL heat inactivated, low-endotoxin FBS. Sterilize by
filtering through 0.22 μm filter.
2. Tissue culture-grade PBS pH 7.4 without calcium chloride or
magnesium chloride.
3. Greiner Bio-One small (18 mm) blade cell scraper.
4. INCYTO C-Chip disposable hemacytometer.
5. 100 mm petri dishes.
6. 50 mL sterile conical tubes.
2.4 Maintenance and
Passage of THP-1 Cells
1. RPMI complete medium (500 mL): 445 mL Roswell Park
Memorial Institute (RPMI) 1640, 5 mL 100Â penicillin
(10,000 I.U./mL) streptomycin (10,000 μg/mL), 50 mL
heat inactivated, low-endotoxin FBS. Sterilize by filtering
through 0.22 μm filter.
2. INCYTO C-Chip disposable hemacytometer.
3. 100 mm petri dishes (or tissue culture flasks).
4. 50 mL sterile conical tubes.
Macrophage Infection Models for Mycobacterium tuberculosis
169