9. Incubate the suspension with mild shaking (100 rpm) for 2 h at
37
C and up to 4 h for THP-1 macrophage cells.
10. Distribute the infected cell suspension equally in sterile 50 mL
centrifuge tubes.
11. Centrifuge at 220 Â g for 10 min.
12. Resuspend in 20 mL of RPMI-FBS supplemented with 50 μg/
mL amikacin. A volume of 20 mL of cell suspension per plate is
required.
13. Incubate the suspension with mild shaking (100 rpm) for 1 h at
37
C. This step is required to kill remaining extracellular
bacteria.
14. Repeat three times the steps 10–12, using 50 mL of RPMIFBS for cell pellet resuspension.
15. Resuspend in 20 mL or 40 mL of RPMI-FBS (to have the cell
type-adapted concentration per well) containing 40 ng/mL
M-CSF, GM-CSF or hM-CSF for primary murine macrophages, murine dendritic cells and human macrophages,
respectively, or no growth factors for cell lines. A volume of
20 mL of cellular suspension per plate is required.
16. Dispense 40 μL (corresponding to 1 Â 10
3 A549, or 2 Â 10
4
RAW264.7, THP-1 macrophages and BMDM, or 4 Â 10
4
BMDC cells/well) of infected cells per well for each assay
plate prepared for compound screening, using a multichannel
pipette or an automatic plate dispenser (see Note 10). If
needed, fill empty wells with medium to limit evaporation
and border effects.
17. Incubate the plates for 5 days at 37
C, 5% CO 2 .
18. Directly add 10 μL of 25 μM SYTO™ 60 into the wells of
384-well assay plate to obtain a final concentration of 5 μM
SYTO™ 60 per well.
Optional step: Perform any staining or any phenotypic
assay following the different detailed steps from the addition
of dye solutions (see Subheadings 3.15–3.24).
19. Seal plates using breathable sealing films.
20. Incubate the plates for 30 min at 37
C with 5% CO 2 .
21. Proceed to image the cells without washing, using the highcontent confocal microscope (see Subheading 3.25). Use 20Â
magnification. Plates cannot be stored.
3.13 Intracellular
Mycobacterial
Replication Assays
for Mutant Screening
The overview diagram of the following steps is shown in Fig. 2c.
1. Prepare bacterial suspensions of mutants at 2 Â 10
6 CFU/mL
(see Subheading 3.1 and Note 18).
678
Nathalie Deboosere et al.
37
C and up to 4 h for THP-1 macrophage cells.
10. Distribute the infected cell suspension equally in sterile 50 mL
centrifuge tubes.
11. Centrifuge at 220 Â g for 10 min.
12. Resuspend in 20 mL of RPMI-FBS supplemented with 50 μg/
mL amikacin. A volume of 20 mL of cell suspension per plate is
required.
13. Incubate the suspension with mild shaking (100 rpm) for 1 h at
37
C. This step is required to kill remaining extracellular
bacteria.
14. Repeat three times the steps 10–12, using 50 mL of RPMIFBS for cell pellet resuspension.
15. Resuspend in 20 mL or 40 mL of RPMI-FBS (to have the cell
type-adapted concentration per well) containing 40 ng/mL
M-CSF, GM-CSF or hM-CSF for primary murine macrophages, murine dendritic cells and human macrophages,
respectively, or no growth factors for cell lines. A volume of
20 mL of cellular suspension per plate is required.
16. Dispense 40 μL (corresponding to 1 Â 10
3 A549, or 2 Â 10
4
RAW264.7, THP-1 macrophages and BMDM, or 4 Â 10
4
BMDC cells/well) of infected cells per well for each assay
plate prepared for compound screening, using a multichannel
pipette or an automatic plate dispenser (see Note 10). If
needed, fill empty wells with medium to limit evaporation
and border effects.
17. Incubate the plates for 5 days at 37
C, 5% CO 2 .
18. Directly add 10 μL of 25 μM SYTO™ 60 into the wells of
384-well assay plate to obtain a final concentration of 5 μM
SYTO™ 60 per well.
Optional step: Perform any staining or any phenotypic
assay following the different detailed steps from the addition
of dye solutions (see Subheadings 3.15–3.24).
19. Seal plates using breathable sealing films.
20. Incubate the plates for 30 min at 37
C with 5% CO 2 .
21. Proceed to image the cells without washing, using the highcontent confocal microscope (see Subheading 3.25). Use 20Â
magnification. Plates cannot be stored.
3.13 Intracellular
Mycobacterial
Replication Assays
for Mutant Screening
The overview diagram of the following steps is shown in Fig. 2c.
1. Prepare bacterial suspensions of mutants at 2 Â 10
6 CFU/mL
(see Subheading 3.1 and Note 18).
678
Nathalie Deboosere et al.