The overall goal of this protocol is to demonstrate the proper techniques for monitoring tumor cell and intracranial tumor growth using in vivo bioluminescence imaging to verify the utility of luciferase modification of GL261 murine tumor cells. This method can help answer key questions in the study of tumor immunology and immunotherapeutic approach. Such as whether the stress modification affects GL261 cell proliferation and grows in vivo.

The main advantage of this technique that luciferase modification of GL261 tumor cells facilitates that noninvasive monitoring of their growth and response to treatment in situ. When the GL261 luciferase cell culture reaches 70%confluency in a T75 flask, harvest the cells by trypsinization. Count the cells and then transfer them to a 24-well plate at graduating densities.

After three hours in a cell culture incubator, open the bioluminescence imaging software and initialize the imaging system. When the camera has cooled to negative 90 degrees Celsius, set the imaging software to Luminescent with a 10-second exposure time and a Medium Binning. Then incubate the cells in 25 microliters of luciferin per well for one minute at room temperature, and place the plate on the imaging station.

Select Acquire to start the imaging. When an image has been successfully obtained, an image window and tool pallet will appear. Click the Regions of Interest Tools and select 6 by 4 from the slit icon.

Cover the area of signal with 6 by 4 slits, and select the pencil icon on the Measurements tab. A window with a table of region of interest measurements as units of photons per second per steradian per square centimeter will appear. To perform an in vitro proliferation assay, harvest the cell cultures as just demonstrated when they reach 70%confluency.

And use a multi-channel pipette to place the tumor cells in 20 wells each of a 96-well plate at 1.5 times 10 to the 3rd cells per 80 microliters of RPMI medium per well. To limit the evaporation of the cell cultures, fill the surrounding empty wells with 100 microliters of fresh RPMI medium. Then, to assess the cellular proliferation, add 20 microliters of MTS reagent to each well of cells.

And incubate the plate at 37 degrees Celsius and 5%carbon dioxide. After three hours, use a microplate reader to measure the absorbance at 490 nanometers. To normalize the absorbance values, divide the values from each day by the corresponding readings obtained on day one.

On the day of implantation, suspend one times 10 to the 5th cells per microliter in HBSS without calcium and magnesium from a 70%confluent tumor-cell culture. Next, after confirming the appropriate level of sedation by toe pinch, shave the top of the head of each experimental animal. Using a cotton tip application, clean each skull with 2%chlorhexidine.

Then apply lubricant to the eyes of each mouse. Now use a sterile scalpel to make a sagittal incision approximately 1 centimeter long over the parietal occipital bone of the first animal. And clean the surface of the skull again with 3%hydrogen peroxide noting the apparent bregma.

Then, using a sterile 25-gauge needle, drill a 3-millimeter hole in the skull to the right of the bregma and just behind the coronal suture. To ensure the appropriate injection depth, use scissors to cut 3 millimeters off the pointed end of a 20-microliter pipette tip, and slide a 26-guage Hamilton syringe into the tip until 3 millimeters of the needle are sticking out of the end of the tip. Insert the needle into the hole in the skull, and slowly inject 3 microliters of 3 times 10 to the 5th of the tumor cells over a one-minute period.

Leave the needle in place for another minute and then slowly withdraw it. Swap the exposed bone with 3%hydrogen peroxide and 2%chlorhexidine solution. Then, after drying the skull with a fresh cotton tip applicator, cut out a 3 square millimeter piece of sterile bone wax and attach the wax to a bare end of a cotton tip applicator.

Cover the injection site with the bone wax. Then use forceps to draw each side of the scalp over the wax. After closing the wound and administering the appropriate post-operative analgesia, repeat the procedure for each experimental animal.

To image the growth of the intracranial tumor, first initialize the imaging station as just demonstrated. Next, after confirming the appropriate level of sedation by tail pinch, set up the imaging system parameters as just demonstrated. With the exception of selecting Auto for the exposure time.

When the imaging system is ready, place the mice onto the imaging station and select Acquire to obtain images of the luciferase expression. When the image has been successfully acquired, select the Region of Interest Tool from the tool pallet and Auto from the circle icon. Encircle the areas of signal to define the regions of interest and then obtain measurements of the regions as units of photons per second, per steradian per square centimeter.

Finally, remove the mice from the imaging chamber, and monitor the tumor-bearing animals until they are fully recovered. In vitro bioluminescence imaging of GL261 cells infected with luciferase-containing lentivirus exhibit a robust luciferase expression similar to the luciferase levels expressed by a positive control U87MG luciferase-expressing human glioblastoma cell line. As expected, uninfected GL261 cells display no background luciferase expression.

Before intracranial implantation, no difference in in vitro growth rates of GL261 luciferase and GL261 cell lines is observed. As serial bioluminescence imaging of intracranial tumor-injected animals demonstrates, GL261 luciferase-expressing tumor cells also exhibit a rapid and consistent growth rate in vivo with no significant difference in survival rates between the experimental tumor-bearing groups observed. Once mastered, this technique can be used to image as many as 25 mice per hour if it is performed properly.

After its development, this technique enhances the ability of bioluminescence imaging to be useful monitoring GL261 tumor growth in vivo furthering the investigation of immunotherapeutic responses in immunocompetent mouse models.