8. Scramble siRNA solution: dilute ON-TARGETplus Non-targeting Control Pool (Horizon Discovery or Dharmacon), for
RNAi experiments in human and mouse cells, or siGENOME
Non-Targeting siRNA Pool #2 (Horizon Discovery) for
human cell screening, with 1Â siRNA buffer to reach a final
stock concentration of 10 or 20 μM. Store stock solutions at
À20
C.
9. Echo (Labcyte) or any other High Precision Automatic Liquid
Dispenser able to accurately dispense volumes of 5–50 μL,
adapted to your 384-well plates (see Note 9).
10. Automatic Plate Washer/Dispenser adapted to your 384-well
plates (see Note 10).
11. Sterile 384-well assay plates with lids, compatible with cell
culture and laser-scanning confocal microscope (see Note 2).
12. Breathable sealing tape for microplates (Corning) or any equivalent adhesive breathable sealing film suitable for your
384-well plates.
13. Laboratory centrifuge for tubes and microplates to achieve
220 Â g.
14. Sterile 50-mL polystyrene reservoirs.
15. Multichannel 50-μL pipette (16 channels).
16. Humidified 37
C incubator maintaining a 5% CO 2
atmosphere.
2.12 Intracellular
Mycobacterial
Replication Assays
for Compound
Screening
N.B. Requirement of a sterile environment, such as a biosafety
cabinet in a dedicated laboratory.
1. See items 9–16 under Subheading 2.11.
2. Dimethylsulfoxide (DMSO): store at room temperature.
3. Borosilicate amber glass vials with hermetic polypropylene
PVDF or PTFE-coated screw caps (see Note 11).
4. Microplate aluminum sealing film (Corning) or any equivalent
adhesive aluminum sealing film suitable for your 384-well
plates.
5. Sterile and ventilated 125 and 250-mL Erlenmeyer.
6. 37
C shaking incubator.
7. Amikacin disulfate salt: store powder at 4
C. Prepare 50 mg/
mL Amikacin stock solution in PBS. Filter-sterilize through a
0.22 μm PTFE membrane and store 100 μL aliquots at À20
C
for up to 1 year. For experiments, freshly dilute the 50 mg/mL
Amikacin solution 1:1000 in RPMI-FBS to obtain a 50 μg/mL
Amikacin solution.
8. 5 mM SYTO™ 60 Red Fluorescent Nucleic Acid Stain solution
(Invitrogen) in DMSO: store at À20
C and protect from light.
For intracellular mycobacterial replication assay, (1) firstly
High-Content Analysis of Host-Pathogen Interactions
657
RNAi experiments in human and mouse cells, or siGENOME
Non-Targeting siRNA Pool #2 (Horizon Discovery) for
human cell screening, with 1Â siRNA buffer to reach a final
stock concentration of 10 or 20 μM. Store stock solutions at
À20
C.
9. Echo (Labcyte) or any other High Precision Automatic Liquid
Dispenser able to accurately dispense volumes of 5–50 μL,
adapted to your 384-well plates (see Note 9).
10. Automatic Plate Washer/Dispenser adapted to your 384-well
plates (see Note 10).
11. Sterile 384-well assay plates with lids, compatible with cell
culture and laser-scanning confocal microscope (see Note 2).
12. Breathable sealing tape for microplates (Corning) or any equivalent adhesive breathable sealing film suitable for your
384-well plates.
13. Laboratory centrifuge for tubes and microplates to achieve
220 Â g.
14. Sterile 50-mL polystyrene reservoirs.
15. Multichannel 50-μL pipette (16 channels).
16. Humidified 37
C incubator maintaining a 5% CO 2
atmosphere.
2.12 Intracellular
Mycobacterial
Replication Assays
for Compound
Screening
N.B. Requirement of a sterile environment, such as a biosafety
cabinet in a dedicated laboratory.
1. See items 9–16 under Subheading 2.11.
2. Dimethylsulfoxide (DMSO): store at room temperature.
3. Borosilicate amber glass vials with hermetic polypropylene
PVDF or PTFE-coated screw caps (see Note 11).
4. Microplate aluminum sealing film (Corning) or any equivalent
adhesive aluminum sealing film suitable for your 384-well
plates.
5. Sterile and ventilated 125 and 250-mL Erlenmeyer.
6. 37
C shaking incubator.
7. Amikacin disulfate salt: store powder at 4
C. Prepare 50 mg/
mL Amikacin stock solution in PBS. Filter-sterilize through a
0.22 μm PTFE membrane and store 100 μL aliquots at À20
C
for up to 1 year. For experiments, freshly dilute the 50 mg/mL
Amikacin solution 1:1000 in RPMI-FBS to obtain a 50 μg/mL
Amikacin solution.
8. 5 mM SYTO™ 60 Red Fluorescent Nucleic Acid Stain solution
(Invitrogen) in DMSO: store at À20
C and protect from light.
For intracellular mycobacterial replication assay, (1) firstly
High-Content Analysis of Host-Pathogen Interactions
657