10. Add 20 μL RPMI-FBS containing or not growth factors (see
step 7).
11. Incubate 3 days at 37
C, 5% CO 2 .
12. Remove the supernatant from plates using a multichannel
pipette.
13. Add 50 μL per well of bacteria at a MOI from 0.5 up to 2 in
RPMI-FBS (see Subheading 3.1 and Note 18). You will need
20 mL of bacterial suspension per plate.
14. Seal plates using breathable sealing films and centrifuge at
220 Â g for 1 min.
15. Incubate for 2 h at 37
C, 5% CO 2 .
16. Remove the breathable sealing films.
17. Wash the cells using an automatic plate washer (see Note 10) or
by carefully pipetting up and down 50 μL of RPMI-FBS in each
well using a multichannel pipette. Finish by removing the
supernatant from the plates.
18. Add 50 μL per well of RPMI-FBS with 50 μg/mL amikacin.
19. Incubate for 1 h at 37
C, 5% CO 2 .
20. Remove the supernatants from the plates using a multichannel
pipette.
21. Repeat step 17 twice.
22. Add 50 μL per well of RPMI-FBS containing or not growth
factors (see step 7).
23. Incubate for 1–5 days at 37
C, 5% CO 2 .
24. Remove the supernatants from the plates using a multichannel
pipette.
25. Perform any staining or any phenotypic assay: follow the
detailed steps from the step describing the addition of staining
solutions (see Subheadings 3.15–3.24).
3.12 Intracellular
Mycobacterial
Replication Assays
for Compound
Screening
The overview diagram of the following steps is shown in Fig. 2b.
The infection procedure (infection of cells in suspension) is detailed
in Fig. 3a.
1. Prepare compound stock solutions at 10 mM by dissolving
pure compounds in 100% DMSO. Use borosilicate amber
glass vials (see Note 11). Store stock solutions at À20
C.
2. Design the source and assay plate layout. An example of a plate
layout is given in Fig. 4.
3. Transfer stock solutions into the 384-well source plates and
prepare intermediate dilutions to reach the final concentration
needed, using micropipettes.
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Nathalie Deboosere et al.
step 7).
11. Incubate 3 days at 37
C, 5% CO 2 .
12. Remove the supernatant from plates using a multichannel
pipette.
13. Add 50 μL per well of bacteria at a MOI from 0.5 up to 2 in
RPMI-FBS (see Subheading 3.1 and Note 18). You will need
20 mL of bacterial suspension per plate.
14. Seal plates using breathable sealing films and centrifuge at
220 Â g for 1 min.
15. Incubate for 2 h at 37
C, 5% CO 2 .
16. Remove the breathable sealing films.
17. Wash the cells using an automatic plate washer (see Note 10) or
by carefully pipetting up and down 50 μL of RPMI-FBS in each
well using a multichannel pipette. Finish by removing the
supernatant from the plates.
18. Add 50 μL per well of RPMI-FBS with 50 μg/mL amikacin.
19. Incubate for 1 h at 37
C, 5% CO 2 .
20. Remove the supernatants from the plates using a multichannel
pipette.
21. Repeat step 17 twice.
22. Add 50 μL per well of RPMI-FBS containing or not growth
factors (see step 7).
23. Incubate for 1–5 days at 37
C, 5% CO 2 .
24. Remove the supernatants from the plates using a multichannel
pipette.
25. Perform any staining or any phenotypic assay: follow the
detailed steps from the step describing the addition of staining
solutions (see Subheadings 3.15–3.24).
3.12 Intracellular
Mycobacterial
Replication Assays
for Compound
Screening
The overview diagram of the following steps is shown in Fig. 2b.
The infection procedure (infection of cells in suspension) is detailed
in Fig. 3a.
1. Prepare compound stock solutions at 10 mM by dissolving
pure compounds in 100% DMSO. Use borosilicate amber
glass vials (see Note 11). Store stock solutions at À20
C.
2. Design the source and assay plate layout. An example of a plate
layout is given in Fig. 4.
3. Transfer stock solutions into the 384-well source plates and
prepare intermediate dilutions to reach the final concentration
needed, using micropipettes.
676
Nathalie Deboosere et al.