12. Incubate for 30 min at room temperature in the dark.
13. Repeat twice steps 2 and 3 with DPBS-FBS.
14. Immediately proceed to image acquisition for F-actin staining
(phalloidins are stable for a short amount of time in 4% formaldehyde fixation buffers), using the high-content confocal
microscope (see Subheading 3.25). Use 20Â or 60Â magnification. Plates used for Hoechst and CellMask Blue staining can
be stored for up to one month, sealed and kept in the dark at
4
C.
3.18 Phagosomal
Acidification Assay
Using LysoTracker
Reagent
This assay can be used in conjunction with M. tuberculosis mutants
for the discovery of new virulence factors, as well as siRNA-silenced
host macrophages or small molecules to determine effectors
involved in phagosomal acidification. M. tuberculosis blocks phagosomal acidification, stabilizing the intra-vacuolar pH around
6.3–6.5. Fluorescent acidotropic probes, such as LysoTracker
(Life Technologies), allow the study of vacuole acidification upon
infection. After cell phagocytosis, live M. tuberculosis resides in
phagosomal
vesicles
escaping
acidification
and
their
co-localization with acidic vacuoles, while heat-killed inactivated
bacteria are targeted by the acidified compartment.
1. Seed 1 Â 10
3 A549 cells, 2 Â 10
4 RAW264.7, THP-1 macrophages, BMDM, or 4 Â 10
4 BMDC (see Subheadings 3.2–3.4
and 3.7–3.9) in 40 μL of appropriate complete medium (with
M-CSF or GM-CSF for BMDM or BMDC) per well in
384-well plates (see Note 21).
2. Incubate plates overnight at 37
C with 5% CO 2 .
3. Remove the supernatant from the plates using a multichannel
pipette.
4. In dedicated wells for positive control (positive phagosomal
acidification or LysoTracker), add 50 μL per well of heat-killed
bacteria at a MOI up to 5.
5. In dedicated wells for cell infection: add 50 μL per well of
bacteria at increasing MOI up to MOI 5 in RPMI-FBS (see
Subheading 3.1 and Note 18).
6. Seal plates using breathable sealing films and centrifuge
220 Â g for 1 min.
7. Incubate plates for 2 h at 37
C with 5% CO 2 .
8. Remove the breathable sealing films.
9. Remove the supernatant from the plates using a multichannel
pipette.
10. Wash cells with 50 μL per well of RPMI-FBS using a multichannel pipette.
High-Content Analysis of Host-Pathogen Interactions
683
13. Repeat twice steps 2 and 3 with DPBS-FBS.
14. Immediately proceed to image acquisition for F-actin staining
(phalloidins are stable for a short amount of time in 4% formaldehyde fixation buffers), using the high-content confocal
microscope (see Subheading 3.25). Use 20Â or 60Â magnification. Plates used for Hoechst and CellMask Blue staining can
be stored for up to one month, sealed and kept in the dark at
4
C.
3.18 Phagosomal
Acidification Assay
Using LysoTracker
Reagent
This assay can be used in conjunction with M. tuberculosis mutants
for the discovery of new virulence factors, as well as siRNA-silenced
host macrophages or small molecules to determine effectors
involved in phagosomal acidification. M. tuberculosis blocks phagosomal acidification, stabilizing the intra-vacuolar pH around
6.3–6.5. Fluorescent acidotropic probes, such as LysoTracker
(Life Technologies), allow the study of vacuole acidification upon
infection. After cell phagocytosis, live M. tuberculosis resides in
phagosomal
vesicles
escaping
acidification
and
their
co-localization with acidic vacuoles, while heat-killed inactivated
bacteria are targeted by the acidified compartment.
1. Seed 1 Â 10
3 A549 cells, 2 Â 10
4 RAW264.7, THP-1 macrophages, BMDM, or 4 Â 10
4 BMDC (see Subheadings 3.2–3.4
and 3.7–3.9) in 40 μL of appropriate complete medium (with
M-CSF or GM-CSF for BMDM or BMDC) per well in
384-well plates (see Note 21).
2. Incubate plates overnight at 37
C with 5% CO 2 .
3. Remove the supernatant from the plates using a multichannel
pipette.
4. In dedicated wells for positive control (positive phagosomal
acidification or LysoTracker), add 50 μL per well of heat-killed
bacteria at a MOI up to 5.
5. In dedicated wells for cell infection: add 50 μL per well of
bacteria at increasing MOI up to MOI 5 in RPMI-FBS (see
Subheading 3.1 and Note 18).
6. Seal plates using breathable sealing films and centrifuge
220 Â g for 1 min.
7. Incubate plates for 2 h at 37
C with 5% CO 2 .
8. Remove the breathable sealing films.
9. Remove the supernatant from the plates using a multichannel
pipette.
10. Wash cells with 50 μL per well of RPMI-FBS using a multichannel pipette.
High-Content Analysis of Host-Pathogen Interactions
683