Imaging on IVIS Spectrum
Janes Lab Protocols
Entered by Sameer Bajikar
November 30, 2015
I.
Before imaging on the IVIS
1. Check the IVIS Spectrum calendar for instrument availability on iLab
2. Use iLab to book the time for the instrument
 Plan for 30 min per one set of mice plus 30 min of set up and clean up
3. Email Dr. Feldman at least 24 hrs ahead of time if you are transferring animals and fax the form to the
CCM
II.
Setting up the IVIS Spectrum
1. If imaging cells in culture, perform steps #2-4 and move to III.
2. Open the software LivingImage
3. Click the “Initialize” button to start up the IVIS Spectrum
 The machine should begin making noise, which is the stage moving into place. The stage will
then begin to heat to 37 degrees Celsius.
4. Set up folders to save the images, and click “Auto-Save-To” and select the folder of interest
5. Once the IVIS Spectrum has been initialized, spray the black plastic matting with Minncare and wipe
with paper towel
6. Remove nose cones from Cavicide cup and dry the cones on a paper towl
7. Place the nose cones into the slots in the IVIS Spectrum cabinet
8. Clean out the isoflurane induction chamber with Cavicide and line the bottom with a clean paper towel
9. Check that the isoflurane tank is filled up by examining the fluid in the meter. The fluid should be
slightly below the upper etched mark. To top off the tank, unscrew the metal stop on the filling
aparatus, unplug the plastic stopper on the glass isoflurane bottle, insert the plastic piece ontop of the
isoflurane bottle into the metal openning on the aparatus and tilt the bottle up. Once the isoflurane fluid
has reached the top etched mark, gently remove the plastic piece and replace the screw top on the
filling aparatus. Replace the plastic plug on the isoflurane bottle
10. Turn on oxygen tank to the left of the IVIS Spectrum cabinet
 The tank should already be set to the appropriate flow rate, so simply turn the tank on by turning
the knob to the right
11. Turn the oxygen flow to the isoflurane tank
III.
Imaging of luciferase activity in cells in culture
1. 24 hours before imaging date, trypsinize and count cells of interest
2. Plate cells in a serial dilution, starting from 40,000 cells per well down to ~312 cells per well in a 96well, black bottom plate
3. Bring cells to the IVIS Spectrum
4. In the BSL2 cabinet, add 10 uL D-luciferin (15 mg/mL) to the media and swirl plate
5. Place plate inside the IVIS Spectrum cabinet
6. Using the software, click on Imaging Wizard. Select open filter, the level of the stage (B is typical for a
96-well plate), auto-exposure, and the duration and interval of imaging (30 min at 2 minute intervals is
our lab standard)
IV.
1.
2.
3.
4.
Imaging luciferase activity in xenograft cells in mice
Place the animals (up to 5) into the isoflurane induction chamber
Turn on the isoflurane on the IVIS isoflurane tank to 2-3% isoflurane
Turn on flow of isoflurane to the induction chamber
Wait until the mice are sedated
 Mice will stop running, typically lay flat on their stomachs or on their side, and breathe slowly
 If tracking mouse weight, wait until after sedation to measure and record weight
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Imaging on IVIS Spectrum
Entered by Sameer Bajikar
Janes Lab Protocols
November 30, 2015
5. Place a sheet of Saran wrap onto the black plastic sheet inside the IVIS Spectrum cabinet
6. Turn on flow of isoflurane to the IVIS Spectrum cabinet
7. Put opthalmaic oinment on the eyes of each sedated mouse
8. Inject mouse with 150 mg/kg D-luciferin in PBS and move the mouse inside the cabinet, placing the
nose of the mouse into the cone in a supine position.
9. Use black masking tape to tape feet to the black mat
10. Using the software, click on Imaging Wizard. Select open filter, the level of the stage (D is typical for
five mice), auto-exposure, and the duration and interval of imaging (30 min at 2 minute intervals is our
lab standard)
V.
1.
2.
3.
4.
5.
6.
VI.
Shut down of IVIS Spectrum
Close LivingImage software, but do not shut down computer
Turn off isoflurane flow to anesthesia chamber and IVIS Spectrum cabinet
Turn on evacuation pump for ~5 min
Return nose cones to Cavicide cup
Spray paper towel with Cavicide and wipe down black imaging mat
Turn off oxygen tank
Bioluminescence image analysis
1. Open up the LivingImage software
2. Open up the image sequence of interest to analyze
3. On the upper left portion of the image sequence, ensure the settings of the image are set to “Radiance”
and not “Counts”
4. On the right menu tool box, select ROI manager and place one ROI on the image sequence
5. Right click on the ROI and set the size of the ROI and lock it into place by clicking the check box
a. The ROI should span one single gland, typically from the base of the abdomen to the
midsection
b. A standard ROI size is 1.5 x 4 inches for our lab’s analysis
6. Place the ROI over a single gland
a. The ROI position will propogate to all the images in the sequence
7. Select measure
8. Copy the data into a separate Excel sheet
9. Record the maximum total photon flux across the image sequence
a. We usually also record the average photon flux for that image as well
b. Record which image contained the peak flux so that one can go back quickly to identify the
image where the data came from
10. Repeat for the remaining glands
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