This method is designed to help scientists to isolate monocyte-derived DCs from the blood and tumors of tumor-bearing mice in order to study their immunostimulatory properties. I think that the main advantage of this procedure compared to other method is that the obtained monocyte-derived DCs better reflect their activation state in the tumor and blood. The hardest thing is probably to make sure that all of your reagents and tools are sterile and that you will not introduce contamination during this procedure.
I've been isolating myeloid cells and dendritic cells from tumors for over 15 years now, and I had the idea of this method when I noticed that different isolation protocols resulted in different patterns of cell activation. Demonstrating the procedure will be Mrs. Santana-Magal, who is a grad student in my lab.
After euthanizing the mice using carbon dioxide inside a laminar flow hood, extract the tumors and place them in RPMI medium without fetal bovine serum. Then, use surgery scissors to slice the tumor into small pieces. Transfer all of the tumor pieces from a single mouse to a 30-milliliter flat-bottom tube containing HBSS buffer supplemented with collagenase IV and DNase I along with the magnetic stir bars.
Incubate the tube with the tumor pieces on a shaker at 37 degrees Celsius with a magnetic stirrer inside at 200 to 400 revolutions per minute for 20 to 30 minutes. Next, filter the cells through a 70-micrometer cell strainer. Then, centrifuge the cells at 400 times g for five to 10 minutes at four degrees Celsius.
After centrifugation, add 1.5 milliliters of density gradient medium stock solution to 8.5 milliliters of HBSS. Then, mix the density gradient medium vigorously. Next, pipette the mixture to the pellet.
Centrifuge the cell suspension at 400 times g for 20 minutes at room temperature. Decant the supernatant after the centrifugation is over. After washing the pelleted cells twice, resuspend in one milliliter of isolation buffer.
Add the cell suspension to 30 microliters of CD11b conjugated magnetic beads. Next, incubate the mixture at four degrees Celsius for 15 minutes. Following centrifugation, remove the supernatant and resuspend the pellet in one milliliter of isolation buffer.
Apply the cell suspension on a pre-washed magnetic column. Next, use three milliliters of isolation buffer to wash the column twice. Then, remove the column from the magnet.
Next, add six milliliters of isolation buffer in the column. Push with a plunger to flush out the magnetically labeled cells in a sterile collection tube. Once again, centrifuge the cells at 400 times g for five to 10 minutes at four degrees Celsius.
After resuspending and staining the cells, sort the cells by gating the small cells, using small side and small forward scatter. After euthanizing the mouse, spray 70%ethanol on it. Then, use surgical scissors to remove the skin covering the heart under the laminar hood.
Next, clean the scissors with ethanol. Wash the cavity with 20-millimolar EDTA HBSS, and use the scissors to cut the right atrium of the heart. Then, use a 10-milliliter syringe with a 25-gauge needle to slowly flush the right ventricle of the heart with 20-millimolar EDTA HBSS.
Then, use a sterile syringe to draw blood from the pleural cavity. Transfer the blood in a tube containing heparan sulfate and EDTA. Next, pipette the blood on the density gradient medium.
Centrifuge the blood cells at 400 times g for 15 minutes at room temperature with low brake. After centrifugation, collect the mononuclear cells in a tube. Dissolve the cells in 10 milliliters of isolation buffer to wash the cells.
Again, centrifuge the cells at 400 times g for five to 10 minutes at four degrees Celsius. To select the CD11b cells, resuspend the cell pellet in one milliliter of isolation buffer. Incubate for 15 minutes with 50 microliters of CD11b conjugated magnetic beads at four degrees Celsius.
Recentrifuge at 400 times g for five to 10 minutes at four degrees Celsius. Then, remove the supernatant. Resuspend the pellet in one milliliter of isolation buffer.
Next, apply the cell suspension on a pre-washed magnetic column. Wash the column with isolation buffer twice. Remove the column from the magnet, and add six milliliters of isolation buffer on the column.
Next, flush out the magnetically labeled cells in a sterile collection tube by pushing the plunger. Centrifuge the cells at 400 times g for five to 10 minutes at four degrees Celsius. Resuspend the cells in isolation buffer and stain with fluorophore-conjugated antibodies.
Sort the cells by gating the small cells using small side and small forward scatter. First, fix the cells in 1.8%buffered paraformaldehyde for 10 minutes at room temperature. Seed the cell suspension in each well of a U-shaped 96-well plate.
Next, add varying dilutions of tumor-binding antibodies in each well. Thereafter, incubate the cells on ice for 15 to 20 minutes. After incubation, wash the cells with 150 microliters of phosphate buffered saline.
Then, centrifuge at 400 times g for five to 10 minutes at four degrees Celsius. Post-centrifugation, expel the supernatant and wash the cells twice. Dissolve the cells in 100 microliters of FACS buffer containing fluorophore-conjugated secondary antibody.
Next, incubate the plate on ice for 15 to 20 minutes. After 15 to 20 minutes, add 200 microliters of FACS buffer to wash the cells. Centrifuge the plate at 400 times g for five to 10 minutes.
Decant the supernatant. Conduct flow cytometry to analyze the tumor binding and to determine the minimal concentration of immunoglobulin G required to coat the cells. Once the cells are coated with minimal immunoglobulin G concentration, add the CFSE-labeled tumor immune complex to the monocyte-derived dendritic cells in a one-to-five ratio.
Then, incubate the mixture for 12 to 16 hours overnight in complete medium. Perform FACS analysis of the monocyte-derived dendritic cell activation. Mean fluorescence intensity is calculated following flow cytometric analysis to study the presence of IgG and M antibodies that bind LMP tumor cells ex vivo.
Results show that the IgG antibodies from C57-Black/6 allogeneic mice bind the tumor cells much stronger than the antibodies from the 129S1 syngeneic mice. Then, the mean fluorescence intensity of the binding capacity of B16F10 tumor cells with the IgG is calculated. IgG obtained from 129S1 allogeneic mice shows 10-fold higher staining intensity with B16F10 tumor in comparison to syngeneic IgG from C57-Black/6 mice.
Next, DIC microscopic images are captured to show the tumor-associated, monocyte-derived dendritic cells from the B16F10 tumors. Here, DIC images are captured to show the inflammatory and patrolling monocyte-derived dendritic cells isolated from the blood of B16F10 tumor-bearing mice. Flow cytometric analysis is also done to compare the activation patterns of monocyte-derived dendritic cells from the spleen and the bone marrow with that of the tumor and blood on incubating with the immune complex.
The result shows increased CD86 and MHC II expression by the bone marrow and splenic dendritic cells with the immune complexes. Then, confocal immunohistochemistry is done, which shows tumor uptake and MHC II expression when the dendritic cells are incubated with the CFSE-labeled tumor immune complex. Once mastered, this procedure can be done in a few hours depending on the number of mice you are using.
While attempting this procedure, it's important to remember to keep all your reagents and tools sterile at all time. Mouse fur is a major source for contaminations. Make sure that none of the hairs are touching your tissues.
We hope that after watching this video, you'll have a good understanding of how to isolate monocyte-derived DCs from mouse blood and tumors and how to subsequently activate them with immune complex cells while avoiding their premature activation.
