9. Wash cells with 50 μL per well of RPMI-FBS using a multichannel pipette.
10. Add 50 μL per well of RPMI-FBS containing 50 μg/mL
amikacin.
11. Incubate plates for 1 h at 37
C with 5% CO 2 .
12. Repeat twice steps 8 and 9.
13. Add 50 μL per well of RPMI-FBS supplemented with M-CSF.
14. In dedicated wells for positive control (oxidative stress positive): add 50 μL per well of RPMI-FBS supplemented with
M-CSF and containing 100 ng/mL LPS and 20 ng/mL IFNγ.
15. Incubate plates for up to 24 h at 37
C with 5% CO 2 .
16. Remove the supernatant from the plates using a multichannel
pipette.
17. Add 50 μL per well of 5 μM CellROX
® Reagent in RPMI-FBS.
18. Incubate plates for 30 min at 37
C.
19. Repeat steps 8 and 9 three times with RPMI-FBS.
20. Add 50 μL per well of 10 μg/mL Hoechst 33342 solution.
21. Incubate for 30 min at 37
C with 5% CO 2 (steps 17 and 20
can be performed simultaneously).
22. Repeat twice steps 8 and 9 with DPBS.
23. Add 50 μL per well of 4% formaldehyde solution used as
fixative solution.
24. Incubate plates for 15 min at room temperature.
25. Repeat twice steps 8 and 9 with DPBS.
26. Proceed to image the cells in 50 μL per well of DPBS-FBS
within 24 h for the CellROX
® Green Reagent and within 2 h
for the CellROX
® Deep Red Reagent, using the high-content
confocal microscope (see Subheading 3.25). Use a 60Â magnification. Plates cannot be stored.
3.22 Oxidative Stress
Assays Using
Intracellular Nitric
Oxide
(NO) Detection Assay
Among all the ROS, production of free radical nitric oxide (NO) is
one antibacterial strategy deployed by macrophages to limit the
proliferation of intracellular pathogens, such as M. tuberculosis. It
was previously shown that M. tuberculosis regulates the amount of
NO through a mechanism involving STAT proteins.
M. tuberculosis-infected human macrophages had a low production
of NO compared to others pathogens. In addition, there is no NO
produced by uninfected human macrophages.
1. Seed and infect cells in 384-well plates and incubate plates for
up to 24 h at 37
C with 5% CO 2 (see Subheading 3.21, steps
1–15).
2. Remove the supernatant from the plates using a multichannel
pipette.
688
Nathalie Deboosere et al.
10. Add 50 μL per well of RPMI-FBS containing 50 μg/mL
amikacin.
11. Incubate plates for 1 h at 37
C with 5% CO 2 .
12. Repeat twice steps 8 and 9.
13. Add 50 μL per well of RPMI-FBS supplemented with M-CSF.
14. In dedicated wells for positive control (oxidative stress positive): add 50 μL per well of RPMI-FBS supplemented with
M-CSF and containing 100 ng/mL LPS and 20 ng/mL IFNγ.
15. Incubate plates for up to 24 h at 37
C with 5% CO 2 .
16. Remove the supernatant from the plates using a multichannel
pipette.
17. Add 50 μL per well of 5 μM CellROX
® Reagent in RPMI-FBS.
18. Incubate plates for 30 min at 37
C.
19. Repeat steps 8 and 9 three times with RPMI-FBS.
20. Add 50 μL per well of 10 μg/mL Hoechst 33342 solution.
21. Incubate for 30 min at 37
C with 5% CO 2 (steps 17 and 20
can be performed simultaneously).
22. Repeat twice steps 8 and 9 with DPBS.
23. Add 50 μL per well of 4% formaldehyde solution used as
fixative solution.
24. Incubate plates for 15 min at room temperature.
25. Repeat twice steps 8 and 9 with DPBS.
26. Proceed to image the cells in 50 μL per well of DPBS-FBS
within 24 h for the CellROX
® Green Reagent and within 2 h
for the CellROX
® Deep Red Reagent, using the high-content
confocal microscope (see Subheading 3.25). Use a 60Â magnification. Plates cannot be stored.
3.22 Oxidative Stress
Assays Using
Intracellular Nitric
Oxide
(NO) Detection Assay
Among all the ROS, production of free radical nitric oxide (NO) is
one antibacterial strategy deployed by macrophages to limit the
proliferation of intracellular pathogens, such as M. tuberculosis. It
was previously shown that M. tuberculosis regulates the amount of
NO through a mechanism involving STAT proteins.
M. tuberculosis-infected human macrophages had a low production
of NO compared to others pathogens. In addition, there is no NO
produced by uninfected human macrophages.
1. Seed and infect cells in 384-well plates and incubate plates for
up to 24 h at 37
C with 5% CO 2 (see Subheading 3.21, steps
1–15).
2. Remove the supernatant from the plates using a multichannel
pipette.
688
Nathalie Deboosere et al.