7. Measure FluoZin-3 mean intensity in corresponding indicator
salt concentration.
8. Plot intensity as median FluoZin-3 intensity to obtain standard
curve representing FluoZin-3 relative intensity in function of
Zinc concentration.
9. Estimate the relative Zinc concentration obtained for infected
cells by converting FluoZin-3 relative intensity per intracellular
bacteria in relative Zinc concentration according to the standard curve.
3.33 Image-Based
Analysis
for Quantification
of Lipid Body
Accumulation [20]
1. Segment host cells, using Hoechst signal and LipidTox intensities to detect nuclei and cell borders, respectively.
2. Detect bacteria using bacteria fluorescence and a “spot” detection algorithm. Define threshold intensity, i.e. pixels with
intensity above the threshold, as small objects considered as
mycobacteria with high intensities compared to the background, and identify as objects within the cytoplasm region.
Remove the background and normalize pixel intensities
between images.
3. Detect lipid droplets (LDs) using LipidTox dye signal
intensities.
4. Select specifically LDs by filtering and refinement using the
algorithm “Find Micronuclei” from Columbus software and
based on size-to-signal intensity and area of LD candidates.
Separate them from out-of-focus and background signal
intensities.
5. Quantify intracellular bacteria as intracellular Mtb area with
number of pixels.
6. Calculate the total area (expressed in pixels) of intracellular
bacteria labeled.
7. Calculate all infection-related parameters. Define “infected”
and “non-infected” populations by using the area of mycobacteria detected within cells as reference criteria. Calculate (1) the
percentage of “LD-positive” cells and (2) the average LD
number per cell.
4 Notes
1. For antibiotic selection: M. tuberculosis H37Rv-GFP and
M. tuberculosis H37Ra-GFP are recombinant strains bearing
an integrative plasmid (based on Ms6) carrying a GFP gene
constitutively expressed from the pBlaF promoter [2] and conferring resistance to hygromycin B. Others strains can be used:
M. tuberculosis H37Rv-RFP is a recombinant strain bearing a
High-Content Analysis of Host-Pathogen Interactions
697
salt concentration.
8. Plot intensity as median FluoZin-3 intensity to obtain standard
curve representing FluoZin-3 relative intensity in function of
Zinc concentration.
9. Estimate the relative Zinc concentration obtained for infected
cells by converting FluoZin-3 relative intensity per intracellular
bacteria in relative Zinc concentration according to the standard curve.
3.33 Image-Based
Analysis
for Quantification
of Lipid Body
Accumulation [20]
1. Segment host cells, using Hoechst signal and LipidTox intensities to detect nuclei and cell borders, respectively.
2. Detect bacteria using bacteria fluorescence and a “spot” detection algorithm. Define threshold intensity, i.e. pixels with
intensity above the threshold, as small objects considered as
mycobacteria with high intensities compared to the background, and identify as objects within the cytoplasm region.
Remove the background and normalize pixel intensities
between images.
3. Detect lipid droplets (LDs) using LipidTox dye signal
intensities.
4. Select specifically LDs by filtering and refinement using the
algorithm “Find Micronuclei” from Columbus software and
based on size-to-signal intensity and area of LD candidates.
Separate them from out-of-focus and background signal
intensities.
5. Quantify intracellular bacteria as intracellular Mtb area with
number of pixels.
6. Calculate the total area (expressed in pixels) of intracellular
bacteria labeled.
7. Calculate all infection-related parameters. Define “infected”
and “non-infected” populations by using the area of mycobacteria detected within cells as reference criteria. Calculate (1) the
percentage of “LD-positive” cells and (2) the average LD
number per cell.
4 Notes
1. For antibiotic selection: M. tuberculosis H37Rv-GFP and
M. tuberculosis H37Ra-GFP are recombinant strains bearing
an integrative plasmid (based on Ms6) carrying a GFP gene
constitutively expressed from the pBlaF promoter [2] and conferring resistance to hygromycin B. Others strains can be used:
M. tuberculosis H37Rv-RFP is a recombinant strain bearing a
High-Content Analysis of Host-Pathogen Interactions
697