16. PBS-Tw (0.05%): 0.05% v/v Tween 80 in PBS, pH 7.4.
17. 1 mL tuberculin syringe (25G Â 5/8
00 ).
18. FACS tubes—5 mL polystyrene round-bottom tube
12 Â 75 mm (Corning Life Sciences, Corning, NY).
19. A flow cytometer with at least a 488 nm laser line. We use a BD
FACSCalibur.
20. FlowJo software (Tree Star, Inc., Ashland, OR).
21. BioSpherix ProOx C21 subchamber controller (BioSpherix,
Parish, NY).
22. BioSpherix C-Chamber (incubator subchamber) (BioSpherix,
Parish, NY).
23. Magnetic stir bars (sterilized).
24. Corning slow speed stirrer (10–150 rpm) (Corning Life
Sciences, Corning, NY).
2.4 Infection of Mice
with Reporter
M. tuberculosis Strains
and Lung Tissue
Collection
1. C57BL/6J or C3HeB/FeJ wild type mice purchased from
Jackson Laboratories or other mice strains as desired.
2. Reporter M. tuberculosis strains (see Subheadings 2.1 and 2.2).
3. 7H9, pH 7.0: Middlebrook 7H9 broth supplemented with
0.2% v/v glycerol and 0.05% v/v Tween 80, pH 7.0 (buffered
with 100 mM 3-(N-Morpholino)-propanesulfonic acid).
4. 7H10 agar.
5. Glycerol.
6. Rodent table-top anesthesia machine.
7. Isoflurane.
8. 2 oz. Whirl-Pak tissue homogenizer bag.
9. Phosphate-buffered saline (PBS), pH 7.4: 1.058 mM
KH 2 PO 4 , 5.6 mM anhydrous Na 2 HPO 4 , 154 mM NaCl.
10. 4% w/v paraformaldehyde, prepared in PBS (PFA).
2.5 Analysis of M.
tuberculosis-Host
Interactions In Vivo,
with Retention of
Spatial Information,
Using Reporter M.
tuberculosis-Infected
Murine Lung Tissue
1. 4% paraformaldehyde-fixed reporter M. tuberculosis-infected
murine lung tissue.
2. A Leica VT1000S fully automatic vibratome (Leica Biosystems,
Buffalo Grove, IL) for thick-sectioning of fixed murine lung
tissue.
3. Stainless steel double edge coated razor blades, 0.004
00 thick
(Electron Microscopy Sciences, Hatfield, PA).
4. Single edge blade, 0.009
00 thick (Electron Microscopy Sciences,
Hatfield, PA).
5. Agarose.
6. 5 mL disposable polystyrene sample beaker.
7. Nutator.
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David Giacalone et al.
17. 1 mL tuberculin syringe (25G Â 5/8
00 ).
18. FACS tubes—5 mL polystyrene round-bottom tube
12 Â 75 mm (Corning Life Sciences, Corning, NY).
19. A flow cytometer with at least a 488 nm laser line. We use a BD
FACSCalibur.
20. FlowJo software (Tree Star, Inc., Ashland, OR).
21. BioSpherix ProOx C21 subchamber controller (BioSpherix,
Parish, NY).
22. BioSpherix C-Chamber (incubator subchamber) (BioSpherix,
Parish, NY).
23. Magnetic stir bars (sterilized).
24. Corning slow speed stirrer (10–150 rpm) (Corning Life
Sciences, Corning, NY).
2.4 Infection of Mice
with Reporter
M. tuberculosis Strains
and Lung Tissue
Collection
1. C57BL/6J or C3HeB/FeJ wild type mice purchased from
Jackson Laboratories or other mice strains as desired.
2. Reporter M. tuberculosis strains (see Subheadings 2.1 and 2.2).
3. 7H9, pH 7.0: Middlebrook 7H9 broth supplemented with
0.2% v/v glycerol and 0.05% v/v Tween 80, pH 7.0 (buffered
with 100 mM 3-(N-Morpholino)-propanesulfonic acid).
4. 7H10 agar.
5. Glycerol.
6. Rodent table-top anesthesia machine.
7. Isoflurane.
8. 2 oz. Whirl-Pak tissue homogenizer bag.
9. Phosphate-buffered saline (PBS), pH 7.4: 1.058 mM
KH 2 PO 4 , 5.6 mM anhydrous Na 2 HPO 4 , 154 mM NaCl.
10. 4% w/v paraformaldehyde, prepared in PBS (PFA).
2.5 Analysis of M.
tuberculosis-Host
Interactions In Vivo,
with Retention of
Spatial Information,
Using Reporter M.
tuberculosis-Infected
Murine Lung Tissue
1. 4% paraformaldehyde-fixed reporter M. tuberculosis-infected
murine lung tissue.
2. A Leica VT1000S fully automatic vibratome (Leica Biosystems,
Buffalo Grove, IL) for thick-sectioning of fixed murine lung
tissue.
3. Stainless steel double edge coated razor blades, 0.004
00 thick
(Electron Microscopy Sciences, Hatfield, PA).
4. Single edge blade, 0.009
00 thick (Electron Microscopy Sciences,
Hatfield, PA).
5. Agarose.
6. 5 mL disposable polystyrene sample beaker.
7. Nutator.
368
David Giacalone et al.