The overall goal of this procedure is to obtain a highly purified mouse lung dendritic cell preparation. This is accomplished by first collecting the lung tissue without including the draining lymph nodes. Then a single cell suspension is obtained by collagenase digestion.
The next step is to enrich the CD 11 C positive cells using a magnetic cell isolation system. Now, the conventional dendritic cell subset is isolated by flow cytometric cell sorting. Ultimately, conventional dendritic cells isolated from mouse lung tissue can be analyzed by flow cytometry and light microscopy.
This method can help answer key questions in the field of immunology, such as what is the involvement of conventional dendritic cell subset in innate immunity and infectious disease in the respiratory tract? Demonstrating the procedure will be Issa Lancelin, a research associate from my laboratory and myself. After euthanizing the mouse begin by cutting and then gently pulling back the outer skin of the peritoneum to expose the thoracic cavity.
Proceed to open the diaphragm by cutting the rib cage to expose both the heart and lungs. Once the heart and lung are exposed, the perfusion procedure can begin. Fill a five milliliter syringe with E-D-T-A-H-B-S-S and connect a 25 gauge needle to the syringe.
Then insert the needle into the right ventricle to prevent leakage During injection. Use forceps to secure the myocardial tissue around the needle. Then gently inject the solution while maintaining a constant pressure.
Accurate perfusion will result in lung inflation and a color change to pink or white to circumvent potential contamination, remove and discard the draining lymph nodes. Then collect the lung tissue, transferring the excised organ to a Petri dish on ice. Fill a gentle max C tube with five milliliters of lung collagenase digestion solution, and then chop the lung into small pieces.
Using a razor blade, transfer the tissue into the tube using a gentle max dissociation. To obtain a single cell suspension, choose program M lung zero one. Then incubate the sample at 37 degrees Celsius for 30 minutes, shaking the tube every five minutes to resuspend the tissue F fragments, proceed with program M lung zero two.
Now transfer the sample to a 15 milliliter conical tube and centrifuge the cells for 10 minutes at 335 times G at four degrees Celsius. Discard the supernatant and add two milliliters of a CK lysing buffer to the pellet for one minute net room temperature to lyce the remaining red blood cells. Then wash the cells with 13 milliliters of cold PBS supplemented with 0.5%BSA.
After discarding the supra natin, again resuspend this pellet in five milliliters of cold PBS plus BSA, and then pass the cell solution through a 100 micrometer nylon mesh. Finally, determine the total cell number by trian blue exclusion. Instead of using a gentle max dissociation, fill a 15 milliliter conical tube with five milliliters collagenase solution per lung.
Add the chopped lung tissue to the tube, incubate the lung tissue for one hour at 37 degrees Celsius. Vortexing the cells every 15 minutes in order to re-spin the tissue fragments. Then disrupt the digestive tissue by passing the sample six to eight times through a three milliliter syringe connected to a 20 gauge needle.
Avoiding making bubbles during the process. First, wash the single cell suspension for 10 minutes at 200 times G at four degrees Celsius and discard the supernatant. Then resuspend the cells in 400 microliters of separation buffer.
Pretend to the eighth total cells block FC mediated unspecific antibody binding using 0.5 micrograms of anti CD 1632 antibody pretended the six cells for 30 minutes at four degrees Celsius. After washing the cells with 10 milliliters of cold separation buffer reus suspend the cells in 400 more microliters of separation buffer. Then add 100 microliters of CD 11 C micro beads, pretend to the eighth total cells, mix well and incubate the cell and bead solution for 15 minutes in the refrigerator.
After removing the unbonded beads with 10 milliliters of cold separation buffer, discard the supernatant and resus suspend the cell pellet in one milliliter of cold separation buffer. Finally, load samples into the AutoMax pro separator and run the P-O-S-S-E-L-D program. To collect the CD 11 C expressing cell fraction.
Incubate the enriched CD 11 C positive cell suspension with anti CD 11 C PEI seven and antibodies directed against the MHC Class Two molecules present on these cells for 30 minutes at four degrees Celsius. After washing the cells with cold PBS plus BSA resuspend the cells and 0.5 milliliters of PBS plus BSA, and pass them through a 40 micrometer nylon mesh filter. Finally, add the purified CD 11 C positive cells to a fax tube for later quantitation in separate blank tubes.
Add 100 microliters of PBS plus BSA to five milliliter collection tubes in order to prevent damage to the purified cells and sort the filtered cell suspension by facts keep the collected cells at a refrigerated temperature throughout the cell sorting process. In these density plots, there are over three times as many conventional dendritic cells found in the spleen as there are in the lung. Further in the lung CD 11 C positive cells include a cell population that are not conventional dendritic cells as represented by the MHC Class two low group of events outside of the conventional dendritic cell gate.
In the left hand side of the figure as seen in this density plot, CD 11 C, magnetic separation of total lung cells increases conventional dendritic cell numbers from about 1.5%of the total cells to about 16.5%However, CD 11 C positive MHC Class two low macro fass still make up over 70%of the total cell population significantly contaminating the purity of the enriched lung conventional dendritic cell subset at this stage of the isolation process. Sorting by fax however, enhances the conventional dendritic cell purity to over 96%as seen in the upper right micro photograph. The CD 11 C high MHC Class two high conventional dendritic cells are rounded cells bearing the typical dendrites, whereas the CD 11 C high MHC class two low cells seen in the bottom right micro photograph exhibit cellular protuberances that are more typical of macrophage morphology.
While attempting this procedure, it's important to remember to handle the lung tissue and the single cell preparation under the indicated temperatures. For this is critical for the preservation of cell viability and conventional dendritic cell yield. After watching this video, you should have a good understanding of how to isolate dendritic cell subsets from the mouse lung.
The isolation of pulmonary dendritic cell subsets represent a very useful tool for gaining insight into the functions of these cells in response to respiratory pathogens and environmental factors that can trigger the host immune response in the lung.
