2. Distribute 20 μL of bacterial suspensions into 384-well assay
plates (see Note 10).
3. Prepare a cellular suspension as previously described (see Subheadings 3.2–3.4 and 3.7–3.10) to have the cell type-adapted
MOI (see Note 18). A volume of 12 mL of cellular suspension
per plate is required.
4. Distribute 30 μL of cellular suspension in the 384-well assay
plates containing the mutants, using an automatic plate dispenser (see Note 10) or a multichannel pipette.
5. Seal plates using breathable sealing films and centrifuge
220 Â g for 1 min.
6. Incubate for 2 h at 37
C with 5% CO 2 .
7. Remove the breathable sealing films.
8. Carefully remove the supernatant from the plates and 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.
9. Add 50 μL per well of RPMI-FBS with 50 μg/mL amikacin.
10. Incubate for 1 h at 37
C, 5% CO 2 .
11. Repeat step 8 twice.
12. Add 50 μL per well of the appropriate complete medium
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.
13. Seal plates using breathable sealing films.
14. Incubate the plates for 5 days at 37
C, 5% CO 2 .
15. Perform any staining or any phenotypic assay following the
different detailed steps from the addition of dye solutions (see
Subheadings 3.15–3.24).
3.14 Intracellular
Mycobacterial Uptake
and Replication
Assays
The infection procedure (infection of adherent cells) is detailed in
Fig. 3b.
1. Seed 1 Â 10
3 A549 cells, 2 Â 10
4 RAW264.7 and THP-1
macrophages and BMDM or 4 Â 10
4 BMDC (see Subheadings
3.2–3.4 and 3.7–3.9) in 40 μL of appropriate complete
medium (with M-CSF or GM-CSF for BMDM or BMDC,
respectively) per well in 384-well plates.
2. Incubate plates overnight at 37
C with 5% CO 2 .
3. Remove the supernatant from the plates using a multichannel
pipette.
High-Content Analysis of Host-Pathogen Interactions
679
plates (see Note 10).
3. Prepare a cellular suspension as previously described (see Subheadings 3.2–3.4 and 3.7–3.10) to have the cell type-adapted
MOI (see Note 18). A volume of 12 mL of cellular suspension
per plate is required.
4. Distribute 30 μL of cellular suspension in the 384-well assay
plates containing the mutants, using an automatic plate dispenser (see Note 10) or a multichannel pipette.
5. Seal plates using breathable sealing films and centrifuge
220 Â g for 1 min.
6. Incubate for 2 h at 37
C with 5% CO 2 .
7. Remove the breathable sealing films.
8. Carefully remove the supernatant from the plates and 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.
9. Add 50 μL per well of RPMI-FBS with 50 μg/mL amikacin.
10. Incubate for 1 h at 37
C, 5% CO 2 .
11. Repeat step 8 twice.
12. Add 50 μL per well of the appropriate complete medium
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.
13. Seal plates using breathable sealing films.
14. Incubate the plates for 5 days at 37
C, 5% CO 2 .
15. Perform any staining or any phenotypic assay following the
different detailed steps from the addition of dye solutions (see
Subheadings 3.15–3.24).
3.14 Intracellular
Mycobacterial Uptake
and Replication
Assays
The infection procedure (infection of adherent cells) is detailed in
Fig. 3b.
1. Seed 1 Â 10
3 A549 cells, 2 Â 10
4 RAW264.7 and THP-1
macrophages and BMDM or 4 Â 10
4 BMDC (see Subheadings
3.2–3.4 and 3.7–3.9) in 40 μL of appropriate complete
medium (with M-CSF or GM-CSF for BMDM or BMDC,
respectively) per well in 384-well plates.
2. Incubate plates overnight at 37
C with 5% CO 2 .
3. Remove the supernatant from the plates using a multichannel
pipette.
High-Content Analysis of Host-Pathogen Interactions
679