24. Resuspend the cell pellet in pre-warmed (37
C) RPMI-FBS
containing 20 ng/mL hM-CSF and adjust to have 1 Â 10
6
cells/mL.
25. Seed 30 mL per 144-cm
2 petri dish (30 Â 10
6 cells per
144-cm
2 petri dish) (see Note 19).
26. Incubate for 3 days at 37
C, 5% CO 2 .
27. Replace the medium with fresh pre-warmed RPMI-FBS containing 20 ng/mL hM-CSF.
28. Optional: To collect non-adhered cells in supernatant, centrifuge the removed supernatant at 330 Â g for 10 min, resuspend
the cell pellet and add in the previous 144-cm
2 petri dish.
29. Incubate cells for 3 days at 37
C, 5% CO 2 .
30. Collect supernatants in a sterile 50-mL centrifuge tube.
31. Add 15 mL per 144-cm
2 petri dish of warm (37
C) DPBS to
rinse and pool with supernatants.
32. Add 20 mL per 144-cm
2 petri dish of pre-warmed BufferPBMC.
33. Incubate cells for 20 min at 37
C, 5% CO 2 .
34. Collect 10 mL of cell suspension in a new sterile 50 mL tube.
35. Flush the 144-cm
2 petri dish with the last 10 mL and pool the
cell suspension.
36. Add 10 mL per 144-cm
2 petri dish of pre-warmed BufferPBMC and repeat step 35.
37. Centrifuge collected supernatants and cells at 330 Â g for
10 min at room temperature and discard the supernatants.
38. Resuspend and pool cells in 45 mL pre-warmed DPBS.
39. Count cells using a counting chamber (expect to obtain
10 Â 10
6 cells per 144 cm
2 petri dish, corresponding to cell
number needed to prepare approximately 50 wells of 384-well
plate).
40. Use for any phenotypic assay (see Subheadings 3.11–3.23).
3.11 Intracellular
Mycobacterial
Replication Assays
for siRNA Screening
The overview diagram of the following steps is shown in Fig. 2a.
The infection procedure (infection of adherent cells) is detailed in
Fig. 3b.
1. Clean all equipment with decontamination reagent for RNase
(RNase AWAY Spray) to work in RNAse-free condition (see
Note 8).
2. Prepare siRNA and scramble siRNA solutions in 384-well
plates to reach the recommended final concentration
(Table 1), by automatic liquid handling (see Note 9).
High-Content Analysis of Host-Pathogen Interactions
673
C) RPMI-FBS
containing 20 ng/mL hM-CSF and adjust to have 1 Â 10
6
cells/mL.
25. Seed 30 mL per 144-cm
2 petri dish (30 Â 10
6 cells per
144-cm
2 petri dish) (see Note 19).
26. Incubate for 3 days at 37
C, 5% CO 2 .
27. Replace the medium with fresh pre-warmed RPMI-FBS containing 20 ng/mL hM-CSF.
28. Optional: To collect non-adhered cells in supernatant, centrifuge the removed supernatant at 330 Â g for 10 min, resuspend
the cell pellet and add in the previous 144-cm
2 petri dish.
29. Incubate cells for 3 days at 37
C, 5% CO 2 .
30. Collect supernatants in a sterile 50-mL centrifuge tube.
31. Add 15 mL per 144-cm
2 petri dish of warm (37
C) DPBS to
rinse and pool with supernatants.
32. Add 20 mL per 144-cm
2 petri dish of pre-warmed BufferPBMC.
33. Incubate cells for 20 min at 37
C, 5% CO 2 .
34. Collect 10 mL of cell suspension in a new sterile 50 mL tube.
35. Flush the 144-cm
2 petri dish with the last 10 mL and pool the
cell suspension.
36. Add 10 mL per 144-cm
2 petri dish of pre-warmed BufferPBMC and repeat step 35.
37. Centrifuge collected supernatants and cells at 330 Â g for
10 min at room temperature and discard the supernatants.
38. Resuspend and pool cells in 45 mL pre-warmed DPBS.
39. Count cells using a counting chamber (expect to obtain
10 Â 10
6 cells per 144 cm
2 petri dish, corresponding to cell
number needed to prepare approximately 50 wells of 384-well
plate).
40. Use for any phenotypic assay (see Subheadings 3.11–3.23).
3.11 Intracellular
Mycobacterial
Replication Assays
for siRNA Screening
The overview diagram of the following steps is shown in Fig. 2a.
The infection procedure (infection of adherent cells) is detailed in
Fig. 3b.
1. Clean all equipment with decontamination reagent for RNase
(RNase AWAY Spray) to work in RNAse-free condition (see
Note 8).
2. Prepare siRNA and scramble siRNA solutions in 384-well
plates to reach the recommended final concentration
(Table 1), by automatic liquid handling (see Note 9).
High-Content Analysis of Host-Pathogen Interactions
673