8. Perform imaging of subsequent groups of mice according to
the same temporal sequence of luciferin administration and
anesthesia procedure.
9. Once the imaging of different experimental groups is complete,
remove all equipment (nose cones, dividers, black sheet) to
decontaminate with a disinfectant. Decontaminate the imaging
tray, check for any residue, close, and disinfect the door.
3.7 Ex Vivo Tissue
Imaging
Blood, urine, and specific tissues such as kidneys, liver, lungs, and
brain from infected mice can be harvested after mice are euthanatized and imaged. This allows to correlate a bioluminescent signal
to the number of bacteria determined by quantitative PCR in a
targeted organ (see Note 10).
1. Euthanize animals in compliance with ethics regulations.
2. Aseptically remove the tissue(s) from one mouse, cut longitudinally (e.g., a kidney), and place each piece of tissue into
individual wells of a 24-well black plate. Keep one well empty
for the blank control of bioluminescence. For urine or blood,
use a 96-well black plate.
3. Immerse tissues in 200 μL of 3 mg/mL D-luciferin per well
including the blank control well. Do not cover the plate.
Transfer the plate to the imaging tray of the IVIS spectrum.
Close the door of the dark box.
4. Select imaging parameters as in step 3 in Subheading 3.4 with
exposure time ¼ 5 min, field of view ¼ C.
5. Click on Acquire on the control screen.
6. After imaging, remove the plate and clean the imaging chamber
and door. Put the plate in a sealed bag, close it, and transfer to
À80
C until the DNA extraction and PCR quantification can
be performed.
3.8 In Vivo Image
Analysis
Bioluminescence imaging is a powerful technology providing valuable information about the spatiotemporal course of the disease in
each animal. The progression of the infection can be monitored by
analyzing and quantifying the light emitted in a selected anatomical
region, at different time points, in the same mouse. By comparing
the level of luminescence in a chosen region, the efficacy of a
potential therapeutic treatment can be evaluated.
1. Select the image acquired in auto-mode acquisition. First,
determine the limits of a region of interest (ROI) that can be
applied to all individuals in the same experiment, using the
mouse with the highest light level.
2. Add ROI for the mouse from the ROI Tools menu. Select the
desired ROI form (see Note 11), adjust the size, and the
position to the targeted region.
156
Fre ´ de ´ rique Vernel-Pauillac and Catherine Werts
the same temporal sequence of luciferin administration and
anesthesia procedure.
9. Once the imaging of different experimental groups is complete,
remove all equipment (nose cones, dividers, black sheet) to
decontaminate with a disinfectant. Decontaminate the imaging
tray, check for any residue, close, and disinfect the door.
3.7 Ex Vivo Tissue
Imaging
Blood, urine, and specific tissues such as kidneys, liver, lungs, and
brain from infected mice can be harvested after mice are euthanatized and imaged. This allows to correlate a bioluminescent signal
to the number of bacteria determined by quantitative PCR in a
targeted organ (see Note 10).
1. Euthanize animals in compliance with ethics regulations.
2. Aseptically remove the tissue(s) from one mouse, cut longitudinally (e.g., a kidney), and place each piece of tissue into
individual wells of a 24-well black plate. Keep one well empty
for the blank control of bioluminescence. For urine or blood,
use a 96-well black plate.
3. Immerse tissues in 200 μL of 3 mg/mL D-luciferin per well
including the blank control well. Do not cover the plate.
Transfer the plate to the imaging tray of the IVIS spectrum.
Close the door of the dark box.
4. Select imaging parameters as in step 3 in Subheading 3.4 with
exposure time ¼ 5 min, field of view ¼ C.
5. Click on Acquire on the control screen.
6. After imaging, remove the plate and clean the imaging chamber
and door. Put the plate in a sealed bag, close it, and transfer to
À80
C until the DNA extraction and PCR quantification can
be performed.
3.8 In Vivo Image
Analysis
Bioluminescence imaging is a powerful technology providing valuable information about the spatiotemporal course of the disease in
each animal. The progression of the infection can be monitored by
analyzing and quantifying the light emitted in a selected anatomical
region, at different time points, in the same mouse. By comparing
the level of luminescence in a chosen region, the efficacy of a
potential therapeutic treatment can be evaluated.
1. Select the image acquired in auto-mode acquisition. First,
determine the limits of a region of interest (ROI) that can be
applied to all individuals in the same experiment, using the
mouse with the highest light level.
2. Add ROI for the mouse from the ROI Tools menu. Select the
desired ROI form (see Note 11), adjust the size, and the
position to the targeted region.
156
Fre ´ de ´ rique Vernel-Pauillac and Catherine Werts