The overall aim of this procedure is to isolate pure and viable primary neuron type two alveolar epithelial cells for subsequent functional and molecular analyses. First, the lung and trachea of a sacrificed mouse are instilled with a solution of DYS displays and then agarose. The lung is then removed after the dispa is allowed to digest the lung.
The tissue is manually disintegrated. The resulting suspension is filtered through a series of meshes of decreasing pore size. Then stained and type two alveolar epithelial cells are isolated by flow cytometric negative selection.
Ultimately, the isolated type two alveolar epithelial cells can be used to perform functional and molecular assays to study their role in respiratory infection and pary autoimmune diseases. This method can help answer key questions in the field of pulmonary immune relation, especially in the context of autoimmune mediad lung diseases and viral infections. Visual demonstration of this method is critical as the organ and cell suspension preparation are difficult to learn.
The overall success of this protocol depends on several important details during these steps For each mouse in the experiment, prewarm a 15 milliliter tube containing four milliliters of DYS bays in a 37 degrees Celsius water bath place, small aliquots of 1%low. Mel Aros in water in a 95 degrees Celsius heating block until liquefied. Then call to 45 degrees Celsius and maintain them at this temperature until they are used next.
To prepare the mouse lung spray a mouse that has been sacrificed by CO2 asphyxiation. With ethanol. Make a long cut along the ventral midline of the body.
Then pull the ventral far skin and carefully cut and remove the peritoneum. After exsanguinating the mouse, carefully puncture and remove the diaphragm and then cut away the ribs. To expose the heart and lung, take special care not to injure the lung with a 26 gauge cannula and a 10 milliliter syringe filled with cold.
PBS puncture the right ventricle of the heart and perfuse the lung with PBS until it is free of blood. Cut and remove the salivary glands to expose the trachea. Also, carefully cut the muscles surrounding the trachea.
Next, insert a 22 gauge indwelling cannula into the trachea. Remove the needle and push the plastic catheter towards the lung. Fix the catheter into place by tying a small piece of yarn around the trachea and catheter.
If desired, prepare a bronchoalveolar lavage fluid sample by flushing the lungs using the catheter inserted in the trachea with PBS or medium. Store the lavage fluid on ice until further analysis. The single most difficult aspect of this procedure is digestion and tissue disintegration.
To ensure the success of this protocol, you have to take care that the complete dispa is filled up in all of the lung lobes and after tissue digestion. You have to disintegrate the tissue Using a two milliliter syringe carefully, still a maximum volume of two milliliters of the dispa prepared earlier into the lung through the catheter so that all lobes are fully expanded. Exchange the syringe for a one milliliter syringe containing 0.5 milliliters of the liquified aros and instill the contents into the lung.
Leave the syringe on the catheter to prevent backflow of the aros and immediately cover the lung with laboratory tissue, paper and ice. Let the aero gel for a couple of minutes. Remove the tissue paper, ice syringe, and catheter.
Then cut the trachea and excise the lung, heart, and thymus from the chest. Rinse the prepared organs in a dish with PBS. Then cut the heart thymus and remaining trachea from the lung and discard them.
Put the lung into the tube containing the remaining two milliliters of displays and incubate it at room temperature for 45 minutes. Alter the incubation to ensure efficient blocking of the FC receptors of phagocytic cells before the staining procedure. Transfer the lung from the displays into a dish containing seven milliliters of one microgram per milliliter, anti CD 1632 antibody and 100 microliters of DNAs in DMEM, using two pairs of forceps completely disintegrate the lung tissue by pulling it apart.
Then incubate it for 10 minutes at room temperature on a rocker set to 200 RPM if desired. The cell suspension can then be stored at four degrees Celsius until the next step. Pipette the cell suspension through a series of nylon meshes.
First, pour the suspension through a 100 micron filter placed on top of a 50 milliliter tube. If pooling samples, pass the cell suspensions from mice of the same group through the same filter. If the filter clogs, exchange it for a new one.
Repeat this process with a 70 micron mesh filter. Take care to rinse each mesh as well as the tubes and dishes thoroughly with DMEM to minimize loss of cells and maximize yield. Next, pull the suspension through a 48 micron mesh filter span onto a plastic or glass ring as shown here.
Collect the flow through in a Petri dish. Then repeat this process with a 30 micron mesh. Depending on the volume, transfer the filtrate to one or more 50 milliliters, tubes and centrifuge for 15 minutes of 160 times G at four degrees Celsius.
After the spin, remove the supinate and resuspend the cell pellet in two milliliters of erythrocyte lysis buffer. Then quickly terminate lysis by addition of 13 milliliters of DMEM medium. Transfer the solution to a 15 milliliter tube and centrifuge for 12 minutes at 160 times G and four degrees Celsius.
Reese has been the cells in three milliliters of a primary antibody cocktail containing antibodies against CD 11 C, CD 11 BF four 80 CD 19, CD 1632, and CD 45. Incubate the cells for 10 minutes in the dark at four degrees Celsius or on ice after the incubation. Wash the cells by filling the tube with DMEM and centrifuge for 12 minutes, a 160 times G and four degrees Celsius.
Re suspend the cells in one milliliter of DMEM and pre-filter through a 50 micron filter into a tube for cell sorting. Count the cells to obtain the total cell number in the lung suspension. Use a 100 micro nozzle for cell sourcing of a EC2 immediately before sourcing.
Mix the cell suspension by vortexing sheath. Fluid pressure and laser remission and detection wavelengths depend on the instrument used for flow cytometric cell sorting as well as the fluorochromes used in the antibody staining procedure. First, create a sideward scatter area versus fluorescence plot and gate on all sideward scatter area High cells negative for the fluorochromes used for staining.
Next, generate sideward scatter area versus forward scatter area and forward scatter height versus forward scatter area and sideward scatter height versus sideward scatter area plots and gate to exclude any doublets or cell aggregates source into a tube containing DMEM. When sourcing lung cell suspensions isolated from healthy mice, the a EC2 gate will typically account for about 42 plus minus 10%of all events. In order to collect the sorted cells centrifuge for 20 minutes at 280 times G and four degrees Celsius.
Re suspend the type two alveolar epithelial cells or a EC2 in culture, media or buffer as required for subsequent analysis. Primary murine type two alveolar epithelial cells were isolated by flow cytometric negative selection as described in this video, analysis of the sorted cells is depicted here. As indicated.
The purity of the isolated cells is roughly 92 plus minus 5%Ciis spins of sorted A EC2 was stained for the surfactant protein CA phase contrast image is shown on the left and immunofluorescence is shown on the right. Nearly 100%of cells were found to be surfactant protein C positive, thus confirming successful isolation of a pure population. To assess the effects of in vivo influenza A virus infection, A EC2 were isolated from C 57 BL six mice that were administered either phosphate buffered saline or influenza A virus PR eight A 34 intranasally one, two or three days earlier.
These plots show lung cell suspensions from an uninfected as well as a three day infected C 57 black six males stained for sorting due to the recruitment of immune cells to the infected lung. The proportion of fluorescence negative side with scatter area high cells is substantially decreased within the cell suspension prepared from the three day infected compared to the uninfected mice. Influenza, A virus nuclear protein expression sorted a C two was analyzed through intracellular staining with a nuclear protein specific antibody.
The histogram shows that a C two isolated from in vivo infected mice yield a specific fluorescence signal after intracellular staining for the influenza A virus, nucleo protein. The table shows the mean fluorescence intensity of the nuclear protein staining the intensity of the signal produced by the intracellular nuclear protein staining of a EC2 isolated from influenza. A virus infected mice increases with the duration of the viral infection to demonstrate the viability of a EC2 isolated from healthy mice and to test their susceptibility to influenza A virus infection.
Influenza, a virus nuclear protein expression was also analyzed in isolated cells infected X vivo as shown here. A substantial proportion of a EC2 expresses the viral nuclear protein six hours post infection. These data are summarized here.
The percentage of a EC2 positive for the intracellular staining of the viral nuclear protein depends on the viral dose used for the ex vivo infection experiment. These results show that the isolated primary neuron, a EC2, are viable and susceptible for viral infection and replication assessed as expression of viral protein seen. While attempting this procedure, it's very important to remember to carefully perform all tissue digestion and disintegration steps.
Also, it's very important to remember to gait stringently on the fluorescence negative and side s getter area high cells. When sorting Following this procedure, other methods like molecular and functional studies can be formed to rule out the role of ular type two tthe third of the lung in the context of viral infections and autoimmune mediad lung diseases.I.
