11. Transfer the samples to new 1.5 mL microcentrifuge tubes and
wash 3 Â 5 min with 500 μL of blocking buffer, on a nutator at
room temperature.
12. Prepare secondary antibodies in blocking buffer, and centrifuge at 13,000 rpm (16,000 Â g) for 5 min, with only the
supernatant taken for use (see Note 14).
13. Move the samples from the last wash ( from step 11) into new
1.5 mL microcentrifuge tubes containing 200 μL of secondary
antibody solution, and incubate for at least 2 h on a nutator at
room temperature.
14. After secondary antibody staining, move the samples a final
time to a new 1.5 mL microcentrifuge tube and wash 3 Â 5 min
with 500 μL of blocking buffer, on a nutator at room
temperature.
15. Mount samples with Vectashield mounting medium (see
Note 15).
16. Use a Leica SP8 spectral confocal microscope to image the
samples (see Notes 5 and 16), with 0.5 μm z-steps. Each
image is most commonly 10–20 μm in depth (see Note 17).
17. Perform reconstruction into 3-dimensions (3D) and analyze
images with Volocity software.
18. Quantify transcriptional reporter signal (e.g., rv2390c’::GFP or
hspX’::GFP) (Fig. 1a) [6, 7] by identifying all objects (bacteria)
in Volocity software via the mCherry signal, which provides
simultaneous measurement of the bacterial volume and the
sum of the GFP signal intensity within each specified bacterial
volume. Calculate GFP/μm
3 values using these measurements.
To ensure accuracy of object calling, verify individually all
“objects” identified by the software, and manually separate
bacterial images as needed.
19. Manually assess the presence/absence of SSB-GFP foci in
Volocity from the 3D-reconstructed or extended focus images
(Fig. 1b) [7]. Here too, use the mCherry signal for identification of bacteria by the software.
20. To ensure data robustness, carry out signal analysis for each
reporter over multiple images, with a minimum of 100 data
points per sample condition.
21. Determine statistical differences between data sets by
performing nonparametric Mann-Whitney tests.
3.6 Preparation of
Single Cell Suspension
from Reporter M.
tuberculosis-Infected
Murine Lung Tissue
1. Harvest lungs from infected mice (see Subheading 3.4, steps
1–10).
2. For each mouse, collect all five lung lobes and mince into small
fragments (~1 mm
3
) in a 6-well plate with scissors.
Exploiting Fluorescent Reporter M. tuberculosis Strains
375
wash 3 Â 5 min with 500 μL of blocking buffer, on a nutator at
room temperature.
12. Prepare secondary antibodies in blocking buffer, and centrifuge at 13,000 rpm (16,000 Â g) for 5 min, with only the
supernatant taken for use (see Note 14).
13. Move the samples from the last wash ( from step 11) into new
1.5 mL microcentrifuge tubes containing 200 μL of secondary
antibody solution, and incubate for at least 2 h on a nutator at
room temperature.
14. After secondary antibody staining, move the samples a final
time to a new 1.5 mL microcentrifuge tube and wash 3 Â 5 min
with 500 μL of blocking buffer, on a nutator at room
temperature.
15. Mount samples with Vectashield mounting medium (see
Note 15).
16. Use a Leica SP8 spectral confocal microscope to image the
samples (see Notes 5 and 16), with 0.5 μm z-steps. Each
image is most commonly 10–20 μm in depth (see Note 17).
17. Perform reconstruction into 3-dimensions (3D) and analyze
images with Volocity software.
18. Quantify transcriptional reporter signal (e.g., rv2390c’::GFP or
hspX’::GFP) (Fig. 1a) [6, 7] by identifying all objects (bacteria)
in Volocity software via the mCherry signal, which provides
simultaneous measurement of the bacterial volume and the
sum of the GFP signal intensity within each specified bacterial
volume. Calculate GFP/μm
3 values using these measurements.
To ensure accuracy of object calling, verify individually all
“objects” identified by the software, and manually separate
bacterial images as needed.
19. Manually assess the presence/absence of SSB-GFP foci in
Volocity from the 3D-reconstructed or extended focus images
(Fig. 1b) [7]. Here too, use the mCherry signal for identification of bacteria by the software.
20. To ensure data robustness, carry out signal analysis for each
reporter over multiple images, with a minimum of 100 data
points per sample condition.
21. Determine statistical differences between data sets by
performing nonparametric Mann-Whitney tests.
3.6 Preparation of
Single Cell Suspension
from Reporter M.
tuberculosis-Infected
Murine Lung Tissue
1. Harvest lungs from infected mice (see Subheading 3.4, steps
1–10).
2. For each mouse, collect all five lung lobes and mince into small
fragments (~1 mm
3
) in a 6-well plate with scissors.
Exploiting Fluorescent Reporter M. tuberculosis Strains
375