The aim of this procedure is to assess pulmonary dendritic cell or DC maturation and migration during the course of Val Boomin induced allergic airway inflammation in mice. This is accomplished by first sensitizing the mice with Val Boomin, followed by airway challenge with fitsy labeled Val Boomin. Next, the lung and the mediastinal lymph nodes are harvested.
Finally, a single cell suspension is prepared from the lung and lymph node tissues. Ultimately, the maturation status and absolute cell number of the Pullman read dendritic cells found in the mediastinal lymph node after FITC Val Boomin challenge can be assessed by flow cytometric analysis. This method can help us answering key questions about pulmonary immune responses such as whether dendretic cells undergo maturation, falling antigen exposure, and whether they migrate to the rating lymph nodes.
And if they do migrate, then what the rate of migration is. Begin by adding freshly prepared Val Boomin solution Dropwise at a one-to-one ratio in a tube of alum while vortexing. Stir the mixture for 30 minutes and then fill a one milliliter syringe with 0.2 milliliters of the mixture.
Using the thumb on forefinger of one hand, grasp the mouse to be injected by the scrap of the neck close to the base of the skull. Then inject the Val Boomin alum solution into the peritoneal cavity of the mouse with the other hand. Seven days after the second injection of Val in alum, hold an anesthetized val in alum sensitized animal in the upright position, and then using sterile pipette tips pipette 100 microliters and freshly prepared val in fxi solution onto the NAS of the mouse in a dropwise fashion.
Begin by attaching a 20 gauge catheter to a one milliliter of syringe. Then after confirming euthanasia of the mouse, open the thoracic cavity of the animal all the way to the neck to expose and cannulate the mouse trachea. It is absolutely critical to lavage the lungs and to profuse the heart to make sure that all the contaminating alveolar macrophages and mononuclear cells that are present in the peripheral blood have been removed.
And make sure to perform these next two steps carefully and of course, with a steady hand. Now administer ice cold PBS with EDTA three times through the catheter to rinse the lower respiratory tract and remove cells from the alveolar spaces. Then after fitting a 10 milliliter syringe with a 20 gauge needle, perfuse the lungs with 10 milliliters of PBS containing heparin via the right ventricle of the heart.
Removal of any remaining blood cells from the lung vasculature is considered complete when the lungs turn white. Next, remove the mediastinal lymph nodes, placing them in a separate dish. Prevent fit se quenching by minimizing the light exposure of the tissue.
Then dissect the perfused lungs and place them in a Petri dish using scissors mince the lungs. Next, digest the lung tissue at 37 degrees Celsius in 250 units per milliliter of collagenase D.After 25 minutes of incubation, add EDTA to the mixture for five minutes to stop the collagenase activity. Finally, pass the fragments of digested lungs through a 100 micron cell strainer and lies the erythrocytes with hypotonic buffer.
Tease apart the mediastinal lymph nodes using fine needles and digest them with collagenase as just demonstrated for the lungs, followed by straining the tissue through a cell strainer following intraperitoneal, sensitization and airway. Val in challenge. The presence of inflammatory cells can be evaluated by hemat, toin and eosin staining in representative lung sections from control.
Saline treated animals, the tissues are devoid of inflammation as demonstrated by the top graphic. In contrast, inflammatory cells are pervasive throughout the lung sections from Val in sensitized and challenged animals as can be seen in the bottom. Graphic, periodic acid shift stain can be used to assess mucus production as shown in this bottom graphic.
The induction of airway allergic inflammation following val in challenge in Val in sensitized mice is confirmed by the presence of mucus. Conversely, a complete absence of mucus production is exhibited by control. Saline treated mice as demonstrated in the top graphic following val in challenge pulmonary dcs undergo maturation and subsequent migration to the draining lymph nodes.
Here a representative analysis of mouse mediastinal lymph nodes shows that a modest but consistent increase in the proportion of CD 11 C positive cells is detected in animal sensitized and challenged with Val Boomin compared to saline treated mice. Moreover, analysis of the absolute cell number of CD 11 C positive CD 11 B positive Val Boomin fits C positive cells from the mediastinal lymph nodes of Val in sensitized and challenged mice shows that a several fold higher increase in DC numbers occurs in challenged mice compared to saline treated animals. After watching this video, we should have a really good understanding of our technique for analyzing pulmonary dendritic cell maturation as well as migration.
And once you really understand this technique, you can go on to modify it to assess pulmonary dendritic cell responses to other pathogens as well as other experimental factors.
