18. At the end of the assay(s), dilute the stored samples (see steps 9,
10, 13, and 17) in 1 mL of PBS-Tw (0.1%) in FACS tubes (at a
dilution of ~1:5 to 1:40 depending on the density of the fixed
bacterial suspension).
19. Just prior to running on the flow cytometer, pass each sample
through a 25G Â 5/8
00 1 mL tuberculin syringe 6–7Â to break
up bacterial clumps.
20. Collect fluorescence data from 10,000 events per sample on a
flow cytometer.
21. Analyze data obtained using FlowJo software to determine
GFP reporter induction.
3.4 Infection of Mice
with Reporter
M. tuberculosis Strains
and Lung Tissue
Collection
1. Grow reporter M. tuberculosis strains to log-phase
(OD 600 ~ 0.6) in 7H9, pH 7.0 medium containing 50 μg/
mL hygromycin for maintenance of the reporter plasmid.
2. Add glycerol to a final concentration of 10% v/v, and store
1 mL aliquots at À80
C.
3. Determine the actual bacterial colony forming units (CFUs) in
the frozen stocks by thawing one vial and transferring the
contents to a 15 mL conical tube, passaging the bacterial
suspension 6–7Â through a 25G Â 5/8
00 1 mL tuberculin
syringe, and plating serial dilutions on 7H10 agar. Count
CFUs after 2–3 weeks.
4. On the day of the mice infection, thaw one vial of each reporter
M. tuberculosis strain as needed, and transfer the contents each
to a 15 mL conical tube.
5. Passage each strains 6–7Â through a 25G Â 5/8
00 1 mL tuberculin syringe.
6. Dilute the stock in PBS-Tw (0.05%), based on the previously
determined CFU/mL (see step 3), to obtain a suspension
containing 1000 CFUs in 35 μL.
7. Plate serial dilutions of this inoculum suspension on 7H10 agar
to verify the bacterial input.
8. Infect 6–8 week old C57BL/6J or C3HeB/FeJ mice (or other
mice as desired) via the intranasal route (see Note 10). Lightly
anesthetize mice with 2% isoflurane delivered via a table-top
anesthesia machine (see Note 11), and instill 35 μL prepared
M. tuberculosis inoculum ( from step 6) down both nares.
9. Place the mice back into a cage and monitor for recovery from
anesthesia.
10. At various time-points post-challenge, sacrifice mice by CO 2
inhalation and harvest the lungs.
11. Place the left lung lobe and accessory right lung lobe in a 2 oz.
Whirl-Pak tissue homogenizer bag containing 500 μL PBS-Tw
(0.05%) and manually homogenize with a roller.
Exploiting Fluorescent Reporter M. tuberculosis Strains
373
10, 13, and 17) in 1 mL of PBS-Tw (0.1%) in FACS tubes (at a
dilution of ~1:5 to 1:40 depending on the density of the fixed
bacterial suspension).
19. Just prior to running on the flow cytometer, pass each sample
through a 25G Â 5/8
00 1 mL tuberculin syringe 6–7Â to break
up bacterial clumps.
20. Collect fluorescence data from 10,000 events per sample on a
flow cytometer.
21. Analyze data obtained using FlowJo software to determine
GFP reporter induction.
3.4 Infection of Mice
with Reporter
M. tuberculosis Strains
and Lung Tissue
Collection
1. Grow reporter M. tuberculosis strains to log-phase
(OD 600 ~ 0.6) in 7H9, pH 7.0 medium containing 50 μg/
mL hygromycin for maintenance of the reporter plasmid.
2. Add glycerol to a final concentration of 10% v/v, and store
1 mL aliquots at À80
C.
3. Determine the actual bacterial colony forming units (CFUs) in
the frozen stocks by thawing one vial and transferring the
contents to a 15 mL conical tube, passaging the bacterial
suspension 6–7Â through a 25G Â 5/8
00 1 mL tuberculin
syringe, and plating serial dilutions on 7H10 agar. Count
CFUs after 2–3 weeks.
4. On the day of the mice infection, thaw one vial of each reporter
M. tuberculosis strain as needed, and transfer the contents each
to a 15 mL conical tube.
5. Passage each strains 6–7Â through a 25G Â 5/8
00 1 mL tuberculin syringe.
6. Dilute the stock in PBS-Tw (0.05%), based on the previously
determined CFU/mL (see step 3), to obtain a suspension
containing 1000 CFUs in 35 μL.
7. Plate serial dilutions of this inoculum suspension on 7H10 agar
to verify the bacterial input.
8. Infect 6–8 week old C57BL/6J or C3HeB/FeJ mice (or other
mice as desired) via the intranasal route (see Note 10). Lightly
anesthetize mice with 2% isoflurane delivered via a table-top
anesthesia machine (see Note 11), and instill 35 μL prepared
M. tuberculosis inoculum ( from step 6) down both nares.
9. Place the mice back into a cage and monitor for recovery from
anesthesia.
10. At various time-points post-challenge, sacrifice mice by CO 2
inhalation and harvest the lungs.
11. Place the left lung lobe and accessory right lung lobe in a 2 oz.
Whirl-Pak tissue homogenizer bag containing 500 μL PBS-Tw
(0.05%) and manually homogenize with a roller.
Exploiting Fluorescent Reporter M. tuberculosis Strains
373