8. Incubate at room temperature for 5 min to lyse red blood cells.
9. Stop red blood cell lysis by adding 10 mL of FACS buffer, and
pellet the suspension via centrifugation at 1500 rpm (450 Â g)
for 10 min (see Note 18).
10. Resuspend the pellet in 10 mL of FACS buffer, determine cell
numbers, and adjust to 5–10 Â 10
6 cells/mL in FACS buffer.
3.7 Flow
Cytometry-Based
Sorting and
Subsequent Confocal
Microscopy-Based
Analyses of Single Cell
Suspensions Derived
from Reporter M.
tuberculosis-Infected
Murine Lung Tissue
1. Incubate lung cell suspensions in 1 mL of FACS buffer with Fc
block (0.5 μg per 10
7 cells) in a 2 mL screw cap tube at room
temperature for 15 min.
2. Centrifuge cells at 3000 rpm (850 Â g) for 5 min.
3. Wash the cells in 1 mL of PBS.
4. Resuspend the washed cell pellet in 1 mL of fixable viability dye
eFluor 506 (diluted 1:1000 in PBS) and incubate for 10 min at
room temperature.
5. Wash the cells again with 1 mL of FACS buffer.
6. Resuspend cells in fluorophore-conjugated flow cytometry
antibodies diluted in 800 μL FACS buffer, and incubate for
30 min in the dark (see Note 19).
7. Remove unbound antibodies by pelleting cells via centrifugation at 3000 rpm (850 Â g) for 5 min, and washing cells once
with 1 mL of FACS buffer.
8. Immediately fix the washed cells with 1.5 mL 4% PFA overnight at room temperature, protected from light.
9. The next day, surface-decontaminate the tubes and remove
from the BSL-3 facility (see Note 20).
10. To prepare fixed cells for flow cytometry, centrifuge the lung
cells at 3000 rpm (850 Â g) for 5 min, and resuspend in sorting
buffer at a concentration of 5 Â 10
6 cells/mL.
11. Pass cell resuspension through a 40 μm cell strainer before
transferring to FACS tubes.
12. Set up the gating strategy to separate major lung phagocytes
that are infected with M. tuberculosis (mCherry-positive cells)
on the BD FACSAria (or other cell sorter being used) and
conduct cell sorting (Fig. 2) (see Note 21).
13. The focus here is on M. tuberculosis-infected host cells, and
thus only mCherry-positive lung phagocytes are collected.
Collect sorted cells into microcentrifuge tubes containing
100 μL sorting buffer, and store at 4
C before confocal
microscopy analyses as needed (see Note 20).
14. Centrifuge the sorted cells at 8000 rpm (6000 Â g) for 5 min.
15. Resuspend the cell pellet in 500 μL of blocking buffer containing DAPI and Alexa Fluor 647-conjugated phalloidin in a
microcentrifuge tube.
Exploiting Fluorescent Reporter M. tuberculosis Strains
377
9. Stop red blood cell lysis by adding 10 mL of FACS buffer, and
pellet the suspension via centrifugation at 1500 rpm (450 Â g)
for 10 min (see Note 18).
10. Resuspend the pellet in 10 mL of FACS buffer, determine cell
numbers, and adjust to 5–10 Â 10
6 cells/mL in FACS buffer.
3.7 Flow
Cytometry-Based
Sorting and
Subsequent Confocal
Microscopy-Based
Analyses of Single Cell
Suspensions Derived
from Reporter M.
tuberculosis-Infected
Murine Lung Tissue
1. Incubate lung cell suspensions in 1 mL of FACS buffer with Fc
block (0.5 μg per 10
7 cells) in a 2 mL screw cap tube at room
temperature for 15 min.
2. Centrifuge cells at 3000 rpm (850 Â g) for 5 min.
3. Wash the cells in 1 mL of PBS.
4. Resuspend the washed cell pellet in 1 mL of fixable viability dye
eFluor 506 (diluted 1:1000 in PBS) and incubate for 10 min at
room temperature.
5. Wash the cells again with 1 mL of FACS buffer.
6. Resuspend cells in fluorophore-conjugated flow cytometry
antibodies diluted in 800 μL FACS buffer, and incubate for
30 min in the dark (see Note 19).
7. Remove unbound antibodies by pelleting cells via centrifugation at 3000 rpm (850 Â g) for 5 min, and washing cells once
with 1 mL of FACS buffer.
8. Immediately fix the washed cells with 1.5 mL 4% PFA overnight at room temperature, protected from light.
9. The next day, surface-decontaminate the tubes and remove
from the BSL-3 facility (see Note 20).
10. To prepare fixed cells for flow cytometry, centrifuge the lung
cells at 3000 rpm (850 Â g) for 5 min, and resuspend in sorting
buffer at a concentration of 5 Â 10
6 cells/mL.
11. Pass cell resuspension through a 40 μm cell strainer before
transferring to FACS tubes.
12. Set up the gating strategy to separate major lung phagocytes
that are infected with M. tuberculosis (mCherry-positive cells)
on the BD FACSAria (or other cell sorter being used) and
conduct cell sorting (Fig. 2) (see Note 21).
13. The focus here is on M. tuberculosis-infected host cells, and
thus only mCherry-positive lung phagocytes are collected.
Collect sorted cells into microcentrifuge tubes containing
100 μL sorting buffer, and store at 4
C before confocal
microscopy analyses as needed (see Note 20).
14. Centrifuge the sorted cells at 8000 rpm (6000 Â g) for 5 min.
15. Resuspend the cell pellet in 500 μL of blocking buffer containing DAPI and Alexa Fluor 647-conjugated phalloidin in a
microcentrifuge tube.
Exploiting Fluorescent Reporter M. tuberculosis Strains
377