3. Middlebrook 7H9 medium supplemented with 10% v/v
OADC and 0.05% v/v Tween-80.
4. Macrophage infection medium (500 mL): 340 mL DMEM
with 4.5 g/L glucose and without sodium pyruvate and Lglutamine, 5 mL 100 mM sodium pyruvate, 5 mL 200 mM
L-glutamine, 50 mL heat inactivated, low-endotoxin FBS,
100 mL L-cell conditioned medium (see Note 2). Sterilize by
filtering through 0.22 μm filter.
5. Basal uptake buffer (250 mL): 250 mL tissue culture-grade
PBS pH 7.4 without calcium chloride or magnesium chloride,
1.125 g glucose, 25 mg calcium chloride, 25 mg magnesium
chloride, 1.25 g bovine serum albumin (BSA), 250 μL 40–50%
cold fish skin gelatin in water. Sterilize by filtering through
0.22 μm filter.
6. Uptake buffer (500 mL): 125 mL basal uptake buffer (see
Subheading 2.6, item 5), 25 mL heat inactivated,
low-endotoxin FBS, 12.5 mL L-cell conditioned medium (see
Note 2), 5 mL 100 mM sodium pyruvate, 332.5 mL DMEM
with 4.5 g/L glucose and without sodium pyruvate and Lglutamine. Sterilize by filtering through 0.22 μm filter.
7. Wash Buffer (500 mL): 457.5 mL DMEM with 4.5 g/L
glucose and without sodium pyruvate and L-glutamine,
25 mL heat inactivated, low-endotoxin FBS, 12.5 mL L-cell
conditioned medium (see Note 2), 5 mL 100 mM sodium
pyruvate. Sterilize by filtering through 0.22 μm filter.
8. Tuberculin syringes: 1 mL TB syringe slip tip with BD PrecisionGlide™ Needle 25 G  5/8 (0.5 mm  16 mm, Becton
Dickinson, Franklin Lakes, NJ).
9. 24-Well tissue culture-treated assay plates.
10. 15 and 50 mL sterile conical tubes.
2.9 Infection of
Primary Bone MarrowDerived Macrophages
with M. tuberculosis in
96- and 384-Well
Plates
1. 96- or 384-well tissue culture-treated assay plates (see Subheading 2.2, item 7).
2. Macrophage infection medium (500 mL) (see Subheading 2.8,
item 4).
3. Basal uptake buffer (250 mL) (see Subheading 2.8, item 5).
4. Uptake buffer (500 mL) (see Subheading 2.8, item 6).
5. Tuberculin syringes (see Subheading 2.8, item 8).
2.10 Quantification
of M. tuberculosis
Survival in
Macrophages
1. Lysis buffer: 0.1% v/v Tween-80 in distilled, deionized water.
Sterilize by filtering through 0.22 μm filter.
2. Dilution buffer: 0.05% v/v Tween-80 in 500 mL of PBS.
Sterilize by filtering through 0.22 μm filter.
Macrophage Infection Models for Mycobacterium tuberculosis
171
OADC and 0.05% v/v Tween-80.
4. Macrophage infection medium (500 mL): 340 mL DMEM
with 4.5 g/L glucose and without sodium pyruvate and Lglutamine, 5 mL 100 mM sodium pyruvate, 5 mL 200 mM
L-glutamine, 50 mL heat inactivated, low-endotoxin FBS,
100 mL L-cell conditioned medium (see Note 2). Sterilize by
filtering through 0.22 μm filter.
5. Basal uptake buffer (250 mL): 250 mL tissue culture-grade
PBS pH 7.4 without calcium chloride or magnesium chloride,
1.125 g glucose, 25 mg calcium chloride, 25 mg magnesium
chloride, 1.25 g bovine serum albumin (BSA), 250 μL 40–50%
cold fish skin gelatin in water. Sterilize by filtering through
0.22 μm filter.
6. Uptake buffer (500 mL): 125 mL basal uptake buffer (see
Subheading 2.6, item 5), 25 mL heat inactivated,
low-endotoxin FBS, 12.5 mL L-cell conditioned medium (see
Note 2), 5 mL 100 mM sodium pyruvate, 332.5 mL DMEM
with 4.5 g/L glucose and without sodium pyruvate and Lglutamine. Sterilize by filtering through 0.22 μm filter.
7. Wash Buffer (500 mL): 457.5 mL DMEM with 4.5 g/L
glucose and without sodium pyruvate and L-glutamine,
25 mL heat inactivated, low-endotoxin FBS, 12.5 mL L-cell
conditioned medium (see Note 2), 5 mL 100 mM sodium
pyruvate. Sterilize by filtering through 0.22 μm filter.
8. Tuberculin syringes: 1 mL TB syringe slip tip with BD PrecisionGlide™ Needle 25 G  5/8 (0.5 mm  16 mm, Becton
Dickinson, Franklin Lakes, NJ).
9. 24-Well tissue culture-treated assay plates.
10. 15 and 50 mL sterile conical tubes.
2.9 Infection of
Primary Bone MarrowDerived Macrophages
with M. tuberculosis in
96- and 384-Well
Plates
1. 96- or 384-well tissue culture-treated assay plates (see Subheading 2.2, item 7).
2. Macrophage infection medium (500 mL) (see Subheading 2.8,
item 4).
3. Basal uptake buffer (250 mL) (see Subheading 2.8, item 5).
4. Uptake buffer (500 mL) (see Subheading 2.8, item 6).
5. Tuberculin syringes (see Subheading 2.8, item 8).
2.10 Quantification
of M. tuberculosis
Survival in
Macrophages
1. Lysis buffer: 0.1% v/v Tween-80 in distilled, deionized water.
Sterilize by filtering through 0.22 μm filter.
2. Dilution buffer: 0.05% v/v Tween-80 in 500 mL of PBS.
Sterilize by filtering through 0.22 μm filter.
Macrophage Infection Models for Mycobacterium tuberculosis
171