8. 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.
9. Blocking buffer: 3% w/v bovine serum albumin (fraction V),
0.1% v/v Triton X-100 in PBS, pH 7.4. Filter sterilize using a
0.22 μm filter.
10. Primary antibodies against host proteins of interest. For example, rabbit anti-inducible nitric oxide synthase (iNOS) (ThermoFisher Scientific, Waltham, MA), rabbit anti-collagen I
(Novus Biologicals, Centennial, CO), and rat anti-CD68
(clone FA-11) (Bio-Rad, Hercules, CA).
11. Secondary antibodies and dyes. For example, Alexa Fluor
514 conjugated goat anti-rabbit IgG, Alexa Fluor 647 conjugated goat anti-rat IgG, Alexa Fluor 647 conjugated phalloidin, and 4
0 ,6-Diamidino-2-Phenylindole, dilactate (DAPI,
dilactate) (ThermoFisher Scientific, Waltham, MA).
12. 75 Â 25 mm microscope slides.
13. 22 Â 22 mm cover glass, No. 1.
14. Vectashield antifade mounting medium (Vector Laboratories,
Burlingame, CA).
15. High vacuum grease (Dow Corning, Auburn, MI).
16. A confocal microscope equipped with at least four laser lines
(405, 488, 594, and 633 nm), and high magnification oil
objectives (40Â, 63Â). We utilize a Leica SP8 spectral confocal
system (see Note 5), with image acquisition via Leica Acquisition Suite X software, and subsequent image analysis carried
out with Volocity software (Quorum Technologies, Ontario,
Canada).
2.6 Preparation
of Single Cell
Suspension from
Reporter M.
tuberculosis-Infected
Murine Lung Tissue
1. Reporter M. tuberculosis-infected murine lung tissue.
2. FACS buffer: 5% v/v FBS in PBS, pH 7.4.
3. Collagenase IV (Worthington Biochemical Corporation,
Lakewood, NJ).
4. ACK (ammonium-chloride-potassium) lysing buffer (Lonza,
Switzerland).
5. 70 μm and 40 μm cell strainers.
2.7 Flow
Cytometry-Based
Sorting and
Subsequent Confocal
Microscopy-Based
Analyses of Single Cell
Suspensions Derived
from Reporter M.
tuberculosis-Infected
Murine Lung Tissue
1. Single cell suspension obtained from reporter M. tuberculosisinfected murine lung tissue.
2. Fluorophore-conjugated antibodies for flow cytometry: Alexa
Fluor 700 rat anti-Ly6G (clone 1A8), PE rat anti-Siglec-F
(clone E50-2440) (BD Biosciences, San Jose, CA), PerCPCyanine5.5 rat anti-CD11b (clone M1/70), Brilliant Violet
421 mouse anti-CD64 (clone X54-5/7.1) (Biolegend, San
Diego, CA), and PE-Cyanine7 rat anti-MerTK (clone
DS5MMER) (eBioscience, San Diego, CA).
Exploiting Fluorescent Reporter M. tuberculosis Strains
369
KH 2 PO 4 , 5.6 mM anhydrous Na 2 HPO 4 , 154 mM NaCl.
9. Blocking buffer: 3% w/v bovine serum albumin (fraction V),
0.1% v/v Triton X-100 in PBS, pH 7.4. Filter sterilize using a
0.22 μm filter.
10. Primary antibodies against host proteins of interest. For example, rabbit anti-inducible nitric oxide synthase (iNOS) (ThermoFisher Scientific, Waltham, MA), rabbit anti-collagen I
(Novus Biologicals, Centennial, CO), and rat anti-CD68
(clone FA-11) (Bio-Rad, Hercules, CA).
11. Secondary antibodies and dyes. For example, Alexa Fluor
514 conjugated goat anti-rabbit IgG, Alexa Fluor 647 conjugated goat anti-rat IgG, Alexa Fluor 647 conjugated phalloidin, and 4
0 ,6-Diamidino-2-Phenylindole, dilactate (DAPI,
dilactate) (ThermoFisher Scientific, Waltham, MA).
12. 75 Â 25 mm microscope slides.
13. 22 Â 22 mm cover glass, No. 1.
14. Vectashield antifade mounting medium (Vector Laboratories,
Burlingame, CA).
15. High vacuum grease (Dow Corning, Auburn, MI).
16. A confocal microscope equipped with at least four laser lines
(405, 488, 594, and 633 nm), and high magnification oil
objectives (40Â, 63Â). We utilize a Leica SP8 spectral confocal
system (see Note 5), with image acquisition via Leica Acquisition Suite X software, and subsequent image analysis carried
out with Volocity software (Quorum Technologies, Ontario,
Canada).
2.6 Preparation
of Single Cell
Suspension from
Reporter M.
tuberculosis-Infected
Murine Lung Tissue
1. Reporter M. tuberculosis-infected murine lung tissue.
2. FACS buffer: 5% v/v FBS in PBS, pH 7.4.
3. Collagenase IV (Worthington Biochemical Corporation,
Lakewood, NJ).
4. ACK (ammonium-chloride-potassium) lysing buffer (Lonza,
Switzerland).
5. 70 μm and 40 μm cell strainers.
2.7 Flow
Cytometry-Based
Sorting and
Subsequent Confocal
Microscopy-Based
Analyses of Single Cell
Suspensions Derived
from Reporter M.
tuberculosis-Infected
Murine Lung Tissue
1. Single cell suspension obtained from reporter M. tuberculosisinfected murine lung tissue.
2. Fluorophore-conjugated antibodies for flow cytometry: Alexa
Fluor 700 rat anti-Ly6G (clone 1A8), PE rat anti-Siglec-F
(clone E50-2440) (BD Biosciences, San Jose, CA), PerCPCyanine5.5 rat anti-CD11b (clone M1/70), Brilliant Violet
421 mouse anti-CD64 (clone X54-5/7.1) (Biolegend, San
Diego, CA), and PE-Cyanine7 rat anti-MerTK (clone
DS5MMER) (eBioscience, San Diego, CA).
Exploiting Fluorescent Reporter M. tuberculosis Strains
369