Immunophenotyping of Orthotopic Homograft (Syngeneic) of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

This message can help answer key questions in Immunophenotyping in Murine models, such as markers used to identify different immunity lineages and aging strategies. The major advantageous of this technique is that the marker and aging stategies, when immunophenotyping, can be apply to a variety of murine models. Monitor the body weight of C57 Black 6 mice using a balance.

Also, monitor the tumor volume of tumor varying donor mice by caliber measurement. Following euthanasia of the animal in a biohazard hood as per protocol, sterilize the skin around the tumor using Iodophor swabs. After surgical removing the tumor as detailed in the text protocol, place the tumor in a petri dish containing 20 milliliter of PBS that has been pre-chilled to 4 degree Celsius.

If there is contaminating blood, transfer the tumor into another Petri dish and wash the tumor with PBS. Cut the tumor in half. Remove any extra skin, vessels, calcification, and necrosis.

Choose only intact pieces of tumor and place them in a sterile 50 milliliter centrifuge tube. Add 20 milliliter of PBS before transporting tube to a separate animal room for pharmacology studies. To prepare for orthotopic implantation, fix a fully anesthetize mouse on an experiment board in the right lateral position.

Disinfect the skin around the spleen with idodine and then de-iodinate with 75%ethyl alcohol. Find the median point of the spleen and make a 1 centimeter vertical incision on the abdomen to expose the spleen. Gently, draw out part of the pancreas tissue under the spleen using flat tip tweezers.

And sutro mouse homograft tumor piece from the C mouse onto the pancreas of the recipient mouse by 9-O bsorbable surgical suture. Close the abdomen with a 6-O silk suture by double seam. Achieve homeostasis by compression.

After finishing tumor implantation, keep the animal in a warm cage as long as no bleeding or tumor tissue leakage occurs. Monitor the animal until they regain sufficient consciousness to maintain sternal recumbency. Return the animal to the animal room after full recovery from anesthesia.

Monitor the tumor varying mice by palpating the abdomen near the spleen and select out the mice bearing orthotopic tumors. For each tumor, prepare one C tube with tumor digestion buffer. Use 3 milliliter of digestion buffer for tumor fragments less than 0.8 grams to ensure the tumor is fully digested.

Label the tumor with the study code, tumor, mouse ID, treatment group, and tumor weight. Collect the tumor from the mouse. Wash the tumor in cold PBS and clean out the tissue attached on the tumor.

Place tumor in digestion media in one well of a sterile 6-well plate. Hold the tumor in place with sterile tweezers or forceps and slice with a scalpel. Slice the tumor well enough to break into smaller pieces.

Now, place the tumor pieces back into the C-tube and use the remaining digestion buffer to wash the plate. Transfer the fluid into the C-tube and place it on ice until digestion. Then switch on the dissociator with heaters.

Place the tumor dissociation C-tube upside down into the sleeve of the vacant position. Adjust the status of tube position from Free to Selected. Choose a dissociation program follow by the selection of the required folder where the list of program is displayed.

After termination of the program, take C-tube off the the dissociator and spin briefly to pallet the sample. Now, re-suspend the samples and put them into a cell strainer above a 50 milliliter tube. Wash the cell through the cell strainer with 10 milliliter of wash buffer to provide a single cell suspension.

After centrifuging tube at 300 G for 5 minutes, discard the supernatant and re-suspend the cells with 5 milliliter of wash buffer. Count viable cells and adjust cell concentration to 1 million cell per tube or per sample. Perform immune panel design, immunostaining and fluorescence minus one control as detail in the text protocol.

Prepare UltraComp beads while the flow instruments is warming up by vortexing them thoroughly before use. Label a separate 12 x 75 millimeter sample tube for each fluorochromes conjugated antibodies. Add 100 microliter of staining buffer to each tube follow by one full drop of the beads.

Add antibodies and perform the staining procedure as described in the text protocol. Then add 0.5 milliliter of staining buffer to each bead pellet and completely re-suspend via vortex. Now, set Flow cytometry PMT voltage per target tissue for the given experiment.

Run the sample through sample through the Flow cytometry for data acquisition by gaining on the singlet bead population per forward scatter and side scatter readings. Set the flow rate around 200 to 300 events per second. Set the appropriate compensation for a given fluorescein FITC conjugation antibodies using an FL1 versus FL1 dot plot.

Place a quadrant gate so that the negative beads are within the lower left quadrant and the positive beads are within the upper or lower right quadrant. Adjust the compensation values until the median fluorescein intensity of each population is approximately equal. Repeat these steps for all tubes.

Now, proceed to acquiring actual stain samples. Run the compensation wizard and save the setting with the format:date, experiment, and your initials. Orthotopic implantation of pancreatic ductal adenocarcinoma resulted in rapid tumor growth similar to that seen in subcutaneous implantation.

Representative hematoxylin and eosin staining images are shown and demonstrate similar growth of subcutaneous and orthotopic implants. Reasonably high viable cells yields were obtain from the tumor sample of both type of implantation. The representative FACS splat shows viable cells from tumor separated from the dead cells and cell debris.

Shown here, is a tumor infiltrating a immune profile comparison of the major and numerator cell population of several key subsets of tumor immune cells. Subcutaneous versus orthotopic homograft of pancreatic cancer are shown. The data clearly shows that the tumor have significantly increase immune cell infiltration as compare with pancreas of healthy mice.

In addition, different percentages of tumor infiltrating immune cell subsets were found in orthotopic versus subcutaneous homografts. For example, there're significantly more B cells in orthotopic than in subcutaneous. While attempting this procedure it is important to remember to prepare all associated materials and reagents in advance.

After watching this video, you should have a good understanding of how to perform FACS analysis for murine models.