3. Add 40 μL per well of pre-warmed (37
C) RPMI-FBS containing 1:400-diluted ROS-ID
® NO Detection Reagent and
Hoechst 0.5 μg/mL.
4. In dedicated wells for negative controls (NO inhibition): add
10 μL per well of a 0.4 mM NO Scavenger suspension, directly
into the pre-diluted ROS-ID
® NO Detection Reagent
(to obtain the recommended final concentration of 80 μM).
5. Incubate plates for 2 h at 37
C with 5% CO 2 .
6. For the last 15–30 min of incubation, in dedicated wells for
positive controls (NO inducer): add 10 μL per well of RPMIFBS containing 5 mM of NO Inducer (L-Arginine) to have a
final concentration of 1 mM.
7. After 2 h total incubation, wash cells twice with 50 μL per well
of 1Â wash buffer (supplied).
8. Remove the supernatant from the plates using a multichannel
pipette.
9. Wash cells with 50 μL per well of RPMI-FBS using a multichannel pipette.
10. Immediately proceed to image the cells in 50 μL per well of
RPMI without red phenol-FBS, using the high-content confocal microscope (see Subheading 3.25). Use 60Â magnification.
Plates cannot be stored.
3.23 Zinc
Poisoning Assay
This assay can be used in conjunction either with siRNAs or with
small molecule screening to determine effectors involved in the
trafficking of zinc within M. tuberculosis-containing phagosomes.
The accumulation of zinc and copper inside phagosomal compartments is a component of the host defense against pathogens. However, virulent mycobacteria can resist this metal-mediated defense
mechanism through the expression of various metal efflux pumps
and transporters. In negative control conditions, the endogenous
zinc is evenly located in the cytoplasm and the intensity of FluoZin3 is minimal. Regions of zinc accumulation (metal deposits) can be
predominantly observed in infected cells, co-localizing with the
presence of M. tuberculosis and with a higher specificity of this
zinc accumulation in bacteria-containing phagosomes than in
infected cell cytoplasm.
1. Seed 2 Â 10
4 Differentiated CD14
+ hPBMCs (see Subheading
3.10) in 40 μL per well of RPMI-FBS supplemented with
50 ng/mL hM-CSF in 384-well plates (see Note 22).
2. Incubate plates overnight at 37
C with 5% CO 2 .
3. Remove the supernatant from the plates using a multichannel
pipette.
4. Infect cells with 50 μL per well of H37Rv-RFP at an MOI of
1 in RPMI-FBS (see Subheading 3.1 and Note 18).
High-Content Analysis of Host-Pathogen Interactions
689
C) RPMI-FBS containing 1:400-diluted ROS-ID
® NO Detection Reagent and
Hoechst 0.5 μg/mL.
4. In dedicated wells for negative controls (NO inhibition): add
10 μL per well of a 0.4 mM NO Scavenger suspension, directly
into the pre-diluted ROS-ID
® NO Detection Reagent
(to obtain the recommended final concentration of 80 μM).
5. Incubate plates for 2 h at 37
C with 5% CO 2 .
6. For the last 15–30 min of incubation, in dedicated wells for
positive controls (NO inducer): add 10 μL per well of RPMIFBS containing 5 mM of NO Inducer (L-Arginine) to have a
final concentration of 1 mM.
7. After 2 h total incubation, wash cells twice with 50 μL per well
of 1Â wash buffer (supplied).
8. Remove the supernatant from the plates using a multichannel
pipette.
9. Wash cells with 50 μL per well of RPMI-FBS using a multichannel pipette.
10. Immediately proceed to image the cells in 50 μL per well of
RPMI without red phenol-FBS, using the high-content confocal microscope (see Subheading 3.25). Use 60Â magnification.
Plates cannot be stored.
3.23 Zinc
Poisoning Assay
This assay can be used in conjunction either with siRNAs or with
small molecule screening to determine effectors involved in the
trafficking of zinc within M. tuberculosis-containing phagosomes.
The accumulation of zinc and copper inside phagosomal compartments is a component of the host defense against pathogens. However, virulent mycobacteria can resist this metal-mediated defense
mechanism through the expression of various metal efflux pumps
and transporters. In negative control conditions, the endogenous
zinc is evenly located in the cytoplasm and the intensity of FluoZin3 is minimal. Regions of zinc accumulation (metal deposits) can be
predominantly observed in infected cells, co-localizing with the
presence of M. tuberculosis and with a higher specificity of this
zinc accumulation in bacteria-containing phagosomes than in
infected cell cytoplasm.
1. Seed 2 Â 10
4 Differentiated CD14
+ hPBMCs (see Subheading
3.10) in 40 μL per well of RPMI-FBS supplemented with
50 ng/mL hM-CSF in 384-well plates (see Note 22).
2. Incubate plates overnight at 37
C with 5% CO 2 .
3. Remove the supernatant from the plates using a multichannel
pipette.
4. Infect cells with 50 μL per well of H37Rv-RFP at an MOI of
1 in RPMI-FBS (see Subheading 3.1 and Note 18).
High-Content Analysis of Host-Pathogen Interactions
689