3. Remove the supernatant from the plates using a multichannel
pipette.
4. In dedicated infected wells: add 50 μL per well of bacteria at an
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.
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 9 and 10.
13. Add 50 μL per well of RPMI-FBS supplemented with M-CSF
or GM-CSF in 384-well plates.
14. In dedicated wells for positive controls (lipid droplet accumulation): add 50 μL per well of RPMI-FBS, supplemented with
M-CSF or GM-CSF and containing 100 ng/mL LPS and
20 ng/mL IFN-γ, or 60 μM Oleic acid, for BMDM or
BMDC, respectively.
15. Incubate plates at 37
C with 5% CO 2 for 2 h up to 4 days.
16. Remove the supernatant from the plates using a multichannel
pipette.
17. Add 50 μL per well of a nucleus staining/cell fixative solution,
corresponding to 10 μg/mL Hoechst 33342 solution diluted
in 4% formaldehyde solution.
18. Incubate for 30 min at room temperature.
19. Repeat twice steps 9 and 10 with DPBS.
20. Add 25 μL per well of 0.5Â LipidTOX neutral lipid stain,
corresponding to the 1000Â LipidTOX neutral lipid stain
diluted 1:2000 in DPBS.
21. Recommended: Seal the plates with opaque plate-sealing film.
22. Incubate the plates at room temperature for at least 15 min.
23. Proceed to image the cells without washing the LipidTOX
stain solution, using the high-content confocal microscope
(see Subheading 3.25). Use 60Â magnification. Plates cannot
be stored and images should be acquired the same day.
High-Content Analysis of Host-Pathogen Interactions
691
pipette.
4. In dedicated infected wells: add 50 μL per well of bacteria at an
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.
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 9 and 10.
13. Add 50 μL per well of RPMI-FBS supplemented with M-CSF
or GM-CSF in 384-well plates.
14. In dedicated wells for positive controls (lipid droplet accumulation): add 50 μL per well of RPMI-FBS, supplemented with
M-CSF or GM-CSF and containing 100 ng/mL LPS and
20 ng/mL IFN-γ, or 60 μM Oleic acid, for BMDM or
BMDC, respectively.
15. Incubate plates at 37
C with 5% CO 2 for 2 h up to 4 days.
16. Remove the supernatant from the plates using a multichannel
pipette.
17. Add 50 μL per well of a nucleus staining/cell fixative solution,
corresponding to 10 μg/mL Hoechst 33342 solution diluted
in 4% formaldehyde solution.
18. Incubate for 30 min at room temperature.
19. Repeat twice steps 9 and 10 with DPBS.
20. Add 25 μL per well of 0.5Â LipidTOX neutral lipid stain,
corresponding to the 1000Â LipidTOX neutral lipid stain
diluted 1:2000 in DPBS.
21. Recommended: Seal the plates with opaque plate-sealing film.
22. Incubate the plates at room temperature for at least 15 min.
23. Proceed to image the cells without washing the LipidTOX
stain solution, using the high-content confocal microscope
(see Subheading 3.25). Use 60Â magnification. Plates cannot
be stored and images should be acquired the same day.
High-Content Analysis of Host-Pathogen Interactions
691