This technique demonstrates a reproducible method to isolate epithelial cells found in the blood and bone marrow of healthy individuals to use for further downstream analysis. This technique allows for the isolation of epithelial cells at a fraction of the cost of automatic circulating tumor cell isolation due to the accessibility of flow cytometry. This method can have implications in developmental and stem cell biology and regenerative therapies.
It can also be used to study wound healing and tissue maintenance in epithelial tissues. The bones are very small and fragile so handle with care to prevent snapping or losing the bones. The bone marrow is relatively stable, so you can work slowly and carefully to ensure proper harvest.
To begin, place the mouse in a 500 milliliter container and add enough iodine to cover half of the mouse. Gently shake and swirl the container to ensure thorough coverage. Then rinse with deionized water.
Perform one more iodine wash and then two washes with 70%ethanol. Place the mouse in a hood and lay it on its back on the tray. Grab the hind legs and pull them apart until a pop is felt.
Make a one centimeter incision on the skin of the mouse near the groin area. Insert a closed pair of scissors into the incision and open the scissors under the skin to separate it from the peritoneum. Cut the skin on the leg around the thigh, the then cut the skin down the leg to expose the muscles and bones.
Before cutting the hind leg, palpate the hip bone to accurately guide the scissors and prevent cuts to the femur where most of the bone marrow is located. Then remove the hind legs by cutting around the hip joint without cutting through the femur. Place the limbs in a tube labeled limbs.
Next, remove the muscles, tissue, and fat along one of the limbs by cutting parallel to the bone using scissors. Then use a scalpel to carefully remove any remaining fat or muscle by performing a scraping motion perpendicular to the bone. After cleaning the bone properly, separate the femur and tibia at the knee.
Then use a scalpel to make cuts on both ends of the femur and tibia where there is no visible bone marrow. Insert the pre-prepared syringe and needle into the bone. If there is resistance, trim the end of the bone until no more resistance is encountered.
Hold the bone firmly above the tube labeled bone marrow and inject the bone marrow harvesting solution into the bone. Then blush out the bone marrow into the tube. Repeat for the other end of the bone to collect the rest of the bone marrow until the bone appears completely white or empty.
Using an empty 10 milliliter syringe and a 20G needle, break the clumps of bone marrow in the tube by drawing the bone marrow up and down in the syringe five to 10 times. Add a 40 micron filter to a clean 50 milliliter centrifuge tube and rinse it with one milliliter of bone marrow harvest solution. Filter the bone marrow through the filter to remove any remaining clumps.
Then label the tube filtered bone marrow. Store the filtered bone marrow in a refrigerator or on ice until use on the same day. Centrifuge the bone marrow cells at 170G for 10 minutes at room temperature.
Vacuum and discard the supernatant. Re-suspend the cells in 10 milliliters of one x red blood cell lysis buffer and incubate for four minutes. Then add 30 milliliters of Dulbecco's phosphate buffered saline to stop the reaction of the lysis buffer.
Centrifuge the cells for eight minutes at 170G at room temperature. Discard the supernatant and re-suspend in 10 to 20 milliliters of staining buffer. First, divide the cells into individually labeled tubes based on the staining panel used, including compensation controls.
Prepare unstained controls and isotope controls to block non-specific staining. For every fluora four, add single stain control and also add fluorescence minus one controls in combination with remaining fluora fours. Next, add antibodies according to the recommended dilution and mix well by flicking the bottom of the tubes.
Preferably use EpCAM conjugated to Phycoerythrin to visualize low populations of cells. Incubate for 30 minutes at four degrees Celsius in the dark. After incubation, bring the tubes to the BSL two hood and add one milliliter of staining buffer to each tube.
Then centrifuge the tubes at 170G at four degrees Celsius for five to 10 minutes. After centrifugation, bring the capped tubes to the BSL two hood and aspirate the supernatant. Re-suspend the cell pellet in one milliliter of staining buffer and flick the bottom of the tubes to mix.
Repeat washing and centrifugation two more times. Finally, re-suspend the palate in 500 microliters of staining buffer before flow cytometry. Add DAPI or dead cell discriminator according to the manufacturer's recommendations.
Using this protocol rare populations of epithelial cells were visualized in the bone marrow of mice. Four to 5%of cells in murine bone marrow were EpCAM positive, regardless of the number of cells counted. In human bone marrow samples two to 5%of the cells were EpCAM positive.
Also, the percentage of cells within each individual donor was consistent as incrementally more cells were counted. In murine blood samples, less than 0.5%of the cells were EpCAM positive. Whereas in human blood samples this number was around 0.3%of the cells.
The control samples showed very few false positive EpCAM positive cells. Further, the cells sorted in the EpCAM positive group showed staining for pan cytokeratin. While those from the EpCAM negative group did not demonstrate the cytokeratin signal.
It is most important to maintain a clean and sterile environment. This will prevent any contamination to the sample. There are several downstream applications that can be used on the isolated epithelial cells to determine their function.
These include RNA sequencing, cell culture in vivo experiments, and immunofluorescence microscopy.
