with a dark-field microscope according to the recommendations for Petroff-Hausser chamber use.
5. Dilute the initial homogenized suspension to the appropriate
infectious dose in DPBS, such that the desired quantity of
leptospires is injected in a 200 μL volume, usually 2 Â 10
7 for
MFLum1 to get a sublethal infection.
6. Transfer the inoculum from the lab to the animal care facility
using a box certified for the transport of infectious substances.
3.3 Mice
Conditioning
The sensitivity of bioluminescence detection is impacted by the
color of the animal’s fur. It is necessary to shave the areas to be
imaged when black mice are used.
1. At least 1 week prior to the experiment, mice are divided into
five individuals per cage, corresponding to the number of
homogeneous experimental groups to evaluate.
2. One day prior to the infection, mice within each group are
individually ear-tagged (see Note 2 and Fig. 3) and, if necessary,
shaved ventrally (see Note 3). Additional marks on the tail
facilitate the manipulation and proper positioning of mice in
the induction box.
3.4 Animal
Inoculation
1. Homogenize the inoculum by inverting the tube and carefully
fill a syringe with 1 mL of the suspension. This allows five mice
to be injected per syringe.
2. Take a cage from the ventilated rack, and deliver 200 μL of
bacterial suspension (or DPBS for control) into the peritoneal
cavity of each identified mouse.
3. Put mice back in a clean cage, and note the experimental
parameters on the identification sheet attached to the cage.
4. Continue the infection with the subsequent groups.
3.5 IVIS Imaging
Preparation
1. Switch on the IVIS imaging station with a personal access code,
and initialize the system using the IVIS Acquisition control
screen as required when connection occurs.
2. Wait for the system to self-calibrate. The heating pad (allowing
for the proper retention of body temperature of the anesthetized mouse) within the dark box is warm as soon as the
temperature indicator becomes green (Fig. 4).
3. Select basic acquisition parameters according to the manufacturer’s instructions:
(a) Imaging mode luminescence with exposure time auto or
5 min, binning medium, F/Stop 1, excitation filter in
block position, emission filter open.
In Vivo Imaging of Bioluminescent Leptospires
153
5. Dilute the initial homogenized suspension to the appropriate
infectious dose in DPBS, such that the desired quantity of
leptospires is injected in a 200 μL volume, usually 2 Â 10
7 for
MFLum1 to get a sublethal infection.
6. Transfer the inoculum from the lab to the animal care facility
using a box certified for the transport of infectious substances.
3.3 Mice
Conditioning
The sensitivity of bioluminescence detection is impacted by the
color of the animal’s fur. It is necessary to shave the areas to be
imaged when black mice are used.
1. At least 1 week prior to the experiment, mice are divided into
five individuals per cage, corresponding to the number of
homogeneous experimental groups to evaluate.
2. One day prior to the infection, mice within each group are
individually ear-tagged (see Note 2 and Fig. 3) and, if necessary,
shaved ventrally (see Note 3). Additional marks on the tail
facilitate the manipulation and proper positioning of mice in
the induction box.
3.4 Animal
Inoculation
1. Homogenize the inoculum by inverting the tube and carefully
fill a syringe with 1 mL of the suspension. This allows five mice
to be injected per syringe.
2. Take a cage from the ventilated rack, and deliver 200 μL of
bacterial suspension (or DPBS for control) into the peritoneal
cavity of each identified mouse.
3. Put mice back in a clean cage, and note the experimental
parameters on the identification sheet attached to the cage.
4. Continue the infection with the subsequent groups.
3.5 IVIS Imaging
Preparation
1. Switch on the IVIS imaging station with a personal access code,
and initialize the system using the IVIS Acquisition control
screen as required when connection occurs.
2. Wait for the system to self-calibrate. The heating pad (allowing
for the proper retention of body temperature of the anesthetized mouse) within the dark box is warm as soon as the
temperature indicator becomes green (Fig. 4).
3. Select basic acquisition parameters according to the manufacturer’s instructions:
(a) Imaging mode luminescence with exposure time auto or
5 min, binning medium, F/Stop 1, excitation filter in
block position, emission filter open.
In Vivo Imaging of Bioluminescent Leptospires
153