16. Remove the supernatant from the plates using a multichannel
pipette.
17. Add 25 μL per well of the substrate loading solution containing 8 μM CCF4-AM in EM buffer-2.5 μM Probenecid.
18. Incubate plates for 2 h at room temperature in the dark (cover
the plate to protect from light and evaporation).
19. Repeat steps 10 and 11 three times with 50 μL per well of EM
buffer-2.5 μM Probenecid.
20. Add 50 μL per well of 4% formaldehyde solution.
21. Incubate for 30 min at room temperature.
22. Repeat twice steps 10 and 11 with DPBS-FBS.
23. Immediately proceed to image the cells in 50 μL per well of
DPBS-FBS, using the high-content confocal microscope (see
Subheading 3.25). Use 60Â magnification. Plates cannot be
stored.
3.21 Oxidative Stress
Assays Using Reactive
Oxygen Species (ROS)
Measurement
Reactive Oxygen Species (ROS) have long been known to be a
component of the killing response of immune cells to microbial
invasion and are highly toxic to bacteria, as they can either directly
destroy DNA, protein, and lipids or indirectly damage nucleic acids
via oxidation of the nucleotide pool. M. tuberculosis infection triggers the generation of reactive oxygen species by host phagocytic
cells (primarily macrophages). However, the survival rate of
M. tuberculosis is highly concentration-dependent of ROS produced by the host immune cells. Exposure of M. tuberculosis to a
low concentration of ROS had no effect on the bacterial cell viability, then the pathogen continued to survive and to replicate in the
host, whereas high concentrations of ROS could be lethal to
M. tuberculosis bacilli.
1. Seed 2 Â 10
4 BMDM (see Subheading 3.8) in 30 μL per well of
RPMI-FBS supplemented with M-CSF in 384-well plates.
2. Incubate plates for 12 h at 37
C with 5% CO 2 .
3. Remove the supernatant from the plates using a multichannel
pipette.
4. In dedicated wells for cell infection: add 50 μL per well of
bacteria at a MOI of 1 or 2 in RPMI-FBS (see Subheading 3.1
and Note 18).
5. Seal plates using breathable sealing films and centrifuge
220 Â g for 1 min at room temperature.
6. Incubate plates for 2 h at 37
C with 5% CO 2 .
7. Remove the breathable sealing films.
8. Remove the supernatant from the plates using a multichannel
pipette.
High-Content Analysis of Host-Pathogen Interactions
687
pipette.
17. Add 25 μL per well of the substrate loading solution containing 8 μM CCF4-AM in EM buffer-2.5 μM Probenecid.
18. Incubate plates for 2 h at room temperature in the dark (cover
the plate to protect from light and evaporation).
19. Repeat steps 10 and 11 three times with 50 μL per well of EM
buffer-2.5 μM Probenecid.
20. Add 50 μL per well of 4% formaldehyde solution.
21. Incubate for 30 min at room temperature.
22. Repeat twice steps 10 and 11 with DPBS-FBS.
23. Immediately proceed to image the cells in 50 μL per well of
DPBS-FBS, using the high-content confocal microscope (see
Subheading 3.25). Use 60Â magnification. Plates cannot be
stored.
3.21 Oxidative Stress
Assays Using Reactive
Oxygen Species (ROS)
Measurement
Reactive Oxygen Species (ROS) have long been known to be a
component of the killing response of immune cells to microbial
invasion and are highly toxic to bacteria, as they can either directly
destroy DNA, protein, and lipids or indirectly damage nucleic acids
via oxidation of the nucleotide pool. M. tuberculosis infection triggers the generation of reactive oxygen species by host phagocytic
cells (primarily macrophages). However, the survival rate of
M. tuberculosis is highly concentration-dependent of ROS produced by the host immune cells. Exposure of M. tuberculosis to a
low concentration of ROS had no effect on the bacterial cell viability, then the pathogen continued to survive and to replicate in the
host, whereas high concentrations of ROS could be lethal to
M. tuberculosis bacilli.
1. Seed 2 Â 10
4 BMDM (see Subheading 3.8) in 30 μL per well of
RPMI-FBS supplemented with M-CSF in 384-well plates.
2. Incubate plates for 12 h at 37
C with 5% CO 2 .
3. Remove the supernatant from the plates using a multichannel
pipette.
4. In dedicated wells for cell infection: add 50 μL per well of
bacteria at a MOI of 1 or 2 in RPMI-FBS (see Subheading 3.1
and Note 18).
5. Seal plates using breathable sealing films and centrifuge
220 Â g for 1 min at room temperature.
6. Incubate plates for 2 h at 37
C with 5% CO 2 .
7. Remove the breathable sealing films.
8. Remove the supernatant from the plates using a multichannel
pipette.
High-Content Analysis of Host-Pathogen Interactions
687