3.25 Image
Acquisition
1. Choose the objective from the microscope. For the determination of the ratio of infected cells, host cell viability or bacterial
load, a 4Â, 10Â or 20Â magnification should be used, as it
allows the recording of a larger number of cells. For the study
of bacterial intracellular trafficking, a higher magnification is
required and a 60Â magnification should be used.
2. Define wells to be read and the number of fields to acquire
from each well. Typically, 4 up to 6 fields are sufficient to allow
the detection of about 1000 cells with a 20Â magnification,
while 10 fields are sufficient to allow the detection of about
100 cells at a 60Â magnification.
3. Set the exposure parameters and acquisition time. Refer to
Table 2 for excitation and emission wavelengths of common
dyes. Cell nuclei and cytoplasm are detected using the blue
channel (DAPI, Hoechst, CellMask Blue Stain) or the far-red
channel (SYTO 60 Red Fluorescent Nucleic Acid Stain).
Plasma membranes are detected using the deep red channel
(CellMask Deep Red Plasma Membrane stain). Bacteria are
detected using the green or red channel, obtained through
the GFP or RFP fluorescence, respectively. Organelle labeling
(“spots”) are detected using the green (LipidTox Green Neutral Lipid Stain, LysoTracker Green Dye), red (LysoTracker
Red Dye) or far-red (LipidTox Far-Red Neutral Lipid Stain,
Alexa Fluor 660 Phalloidin Stain) channels (Fig. 5a). Bacterial
escape can be observed using simultaneously the green (CCF4AM) and the blue channel (CCF4). Molecules or pH change
involved in host defense can be detected using the green channel (CellROX
® Green Oxidative Stress Reagent, FluoZin™-3AM cell permeant) or red channel (Amine-reactive pHrodo™
Red NHS Ester) or far-red channel (CellROX
® Deep Red
Oxidative Stress Reagent, ROS-ID
® Red NO Detection
Reagent).
4. Proceed with plate reading and collecting the images.
3.26 Image-Based
Analysis for Cell
Detection
and M. tuberculosis
Intracellular
Replication
These read-outs can be used for compound screening to identify
bacteriostatic activities of intracellular active anti-TB compounds,
as well as to determine their range of active concentrations by doseresponse. We show the effect of several concentrations of rifampicin
on M. tuberculosis growth within RAW264.7 after 5 days infection
(Fig. 6). Moreover, compound cytotoxic effects can be quantified
simultaneously determined by the number of detected cells
(Fig. 6a). For screening purposes, the total number of infected
cells, the total area of bacteria in infected cells (expressed in pixels)
and the ratio of infected cells (Fig. 6a, b) are the most useful
parameters to determine the extent of infection. In typical kinetics
of M. tuberculosis infection, the increase in the percentage of
infected cells and the expansion of the average bacterial area per
infected cell can be monitored over time.
692
Nathalie Deboosere et al.
Acquisition
1. Choose the objective from the microscope. For the determination of the ratio of infected cells, host cell viability or bacterial
load, a 4Â, 10Â or 20Â magnification should be used, as it
allows the recording of a larger number of cells. For the study
of bacterial intracellular trafficking, a higher magnification is
required and a 60Â magnification should be used.
2. Define wells to be read and the number of fields to acquire
from each well. Typically, 4 up to 6 fields are sufficient to allow
the detection of about 1000 cells with a 20Â magnification,
while 10 fields are sufficient to allow the detection of about
100 cells at a 60Â magnification.
3. Set the exposure parameters and acquisition time. Refer to
Table 2 for excitation and emission wavelengths of common
dyes. Cell nuclei and cytoplasm are detected using the blue
channel (DAPI, Hoechst, CellMask Blue Stain) or the far-red
channel (SYTO 60 Red Fluorescent Nucleic Acid Stain).
Plasma membranes are detected using the deep red channel
(CellMask Deep Red Plasma Membrane stain). Bacteria are
detected using the green or red channel, obtained through
the GFP or RFP fluorescence, respectively. Organelle labeling
(“spots”) are detected using the green (LipidTox Green Neutral Lipid Stain, LysoTracker Green Dye), red (LysoTracker
Red Dye) or far-red (LipidTox Far-Red Neutral Lipid Stain,
Alexa Fluor 660 Phalloidin Stain) channels (Fig. 5a). Bacterial
escape can be observed using simultaneously the green (CCF4AM) and the blue channel (CCF4). Molecules or pH change
involved in host defense can be detected using the green channel (CellROX
® Green Oxidative Stress Reagent, FluoZin™-3AM cell permeant) or red channel (Amine-reactive pHrodo™
Red NHS Ester) or far-red channel (CellROX
® Deep Red
Oxidative Stress Reagent, ROS-ID
® Red NO Detection
Reagent).
4. Proceed with plate reading and collecting the images.
3.26 Image-Based
Analysis for Cell
Detection
and M. tuberculosis
Intracellular
Replication
These read-outs can be used for compound screening to identify
bacteriostatic activities of intracellular active anti-TB compounds,
as well as to determine their range of active concentrations by doseresponse. We show the effect of several concentrations of rifampicin
on M. tuberculosis growth within RAW264.7 after 5 days infection
(Fig. 6). Moreover, compound cytotoxic effects can be quantified
simultaneously determined by the number of detected cells
(Fig. 6a). For screening purposes, the total number of infected
cells, the total area of bacteria in infected cells (expressed in pixels)
and the ratio of infected cells (Fig. 6a, b) are the most useful
parameters to determine the extent of infection. In typical kinetics
of M. tuberculosis infection, the increase in the percentage of
infected cells and the expansion of the average bacterial area per
infected cell can be monitored over time.
692
Nathalie Deboosere et al.