The overall goal of this procedure is to evaluate the ability of poly anhydride nanoparticles to activate purified mirroring alveolar macrophages. First, the trachea of the mouse is surgically exposed and along lavage is performed to harvest cells from the respiratory tract. Alveolar macrophages are then spun down to isolate them and cultured overnight.
A poly anhydride nanoparticle suspension is then prepared and added to the alveolar macrophages. The macrophages are incubated to allow for activation after 48 hours. Cell surface marker expression is analyzed by flow cytometry.
Analysis of the data enables identification of formulations of Polly Anhydride nanoparticles capable of activating mirroring alveolar macrophages. This metal can help to answer questions in the fields of biomaterials and immunology. Specifically, we can identify biomaterials with intrinsic adjuvant activity as evidenced by the ability to activate the engine presenting cells, including alveolar macrophages.
We first had the idea to use this method in our research when we discovered that a poly anhydride nano vaccine platform against respiratory pathogens provided complete protection against bacterial pneumonia. In this present work, we sought to investigate the specific interactions of the vaccine platform with respiratory immune cells. Begin this procedure by placing a euthanized mouse on its back.
Spray the mouse with 70%ethanol, although a six to eight week old C 57 BL six mouse is used here, this technique can be performed on any strain of mouse. Locate the sternum and at a 30 degree angle from the abdomen, insert a 23 gauge needle with a one milliliter syringe. About 50 millimeters into the thoracic cavity, collect 0.4 to one milliliter of blood Generally in the wheels.
Performing this procedure will struggle getting an off sale for their experiments. That's why it's really important to perform carefully as demonstrated here. To perform the lavage, turn the mouse onto its abdomen and spray it with 70%ethanol.
Then pinch the skin about halfway down the spine using dissection scissors that have been sterilized with 70%ethanol. Make a small incision through the skin. Grab the skin on both sides of the incision and pull in opposite directions along the cranial coddle axis of the mouse.
Once the muscles and glands of the neck are exposed, carefully cut muscle tissue. Then using forceps. Pull the tissue toward the anterior of the mouse to expose the larynx, trachea, and esophagus.
Avoid cutting the jugular vein or carotid artery. The trachea is identifiable as the tissue with cartilaginous rings around it. With the forceps delicately, grasp the trachea and gently lift and separate it from the esophagus, which is located on the dorsal side.
Fill a one cc syringe fitted with a tomcat catheter with sterile PBS. Next at a 45 degree angle. Make a small incision in the trachea anterior to the forceps, but posterior to the larynx.
Avoid completely transecting the trachea, which will prevent it from retracting into the body cavity. Using forceps, hold the trachea and gently insert the catheter about 20 millimeters into the trachea incision site. Then grip the trachea that is around the catheter tube to free the other hand while externally massaging the chest.
Use a one milliliter syringe fitted with the catheter to gently infuse 0.75 milliliters of room temperature sterile PBS into the lungs. Then gently aspirate the PBS back into the syringe. Repeat this process three times.
Disconnect the syringe from the catheter and dispense the lavage fluid into a 15 milliliter centrifuge tube. Fill the syringe with 0.75 milliliters of fresh PBS and reconnect the syringe to the catheter and repeat the lavage. Fluid recovery may be partial, so less volume may be obtained in the volume infused.
If cells will be collected from more than one mouse. Place the 15 milliliter tube with the harvested cells on ice until all lung lavages have been performed. To enrich the population of alveolar macrophages in the harvested cells, centrifuge the lung lavage fluid at 250 times G for 10 minutes at four degrees Celsius.
Using sterile technique, decant the supernatant into an appropriate waste container Inside a biosafety cabinet. Resus suspend the cell pellet by gently rigging the centrifuge tube across the top of a test tube rack. Add one milliliter of complete alveolar macrophage medium to resuspend the cells to enumerate the cells.
Pipette 20 microliters of cells into 10 milliliters of isotone two diluent in a culture counter vial. Add three to four drops of zog globin, two lytic reagent into the vial and gently inverted three to four times. Then load the sample vial into a colter particle counter Z one counter.
Be sure to program the counter with the dilution factor of two times 10 to the fourth to display the cell concentration as a number of cells per milliliter. Once the cell concentration has been determined, dilute the cells to 2.5 times 10 to the fifth per milliliter with complete alveolar macrophage medium in a 15 milliliter tube. Then dispense two milliliters of the solution into each well of a six well tissue culture dish.
To enrich the alveolar macrophages, incubate the culture dishes in a 37 degrees Celsius humidified incubator with a 5%CO2 atmosphere for six hours. During the incubation, the macrophages will adhere to the bottom of the well following the incubation, gently aspirate the supernatant taking care not to disrupt the adherent cells. Then rinse the wells using sterile calcium and magnesium free PBS.
To remove non-adherent cells and debris, add two milliliters of complete alveolar macrophage medium to each. Well then incubate the cells at 37 degrees Celsius overnight. After this enrichment step, approximately 87%of the cells will be positive for the macrophage marker CD 11 B and F four 80.
Once the alveolar macrophage population has been enriched, the next step of the procedure is to add poly anhydride nanoparticles first fabricate nanoparticles via poly anhydride antis solvent nano encapsulation. In this process, the polymer is dissolved in methylene chloride and precipitated in pentane. Weigh out the appropriate amount of poly anhydride nanoparticles using sterilized whey paper.
Here, 125 micrograms of particles are added for each milliliter of culture. Add the particles to a 1.5 milliliter micro centrifuge tube, resuspend the nanoparticles in the appropriate amount of ice cold, complete alveolar macrophage medium, and place it on ice. Next, you sterilize the microtip of a benchtop sonicate.
Spray it with 70%ethanol and wipe with a sterile kim wipe while keeping the tube on ice. Sonicate at 10 to 20 joules for 30 to 45 seconds. Observe the nanoparticle suspension macroscopically if the particles are not sufficiently dispersed.
Place the suspension on ice for one to two minutes to ensure that the nanoparticles are below the glass transition temperature of the polymer. Then sonicate again for 30 to 45 seconds. The intent of the sonication step is to adequately disperse the nanoparticles as the 50 50 CCP tag.
CPH nanoparticles tend to aggregate when exposed to an aqueous environment. However, some small aggregates of nanoparticles may still remain in the suspension. The nanoparticle suspension should be kept on ice and used immediately to prevent premature surface erosion or aggregation.
Remove the culture dishes containing the alveolar macrophages from the incubator and place them in the biosafety cabinet. Tilt the plate taking care not to disturb the A adherent cells and pipette off the amount of medium that will be added to the nanoparticle suspension Vortex. The poly anhydride nanoparticle suspension briefly then pipette up to 0.3 milliliters of the nanoparticle suspension into the appropriate wells.
Again, taking care not to disturb the adherent cells. The particles will settle to the bottom of the well and contact the cells. Proceed with subsequent wells and desired treatment groups.
Include positive and negative control groups such as medium only, and 200 nanograms per milliliter. Lipopolysaccharide to designated wells, then incubate at 37 degrees Celsius for 48 hours. Following the incubation, the alveolar activation can be assessed by a flow cytometry as described in the accompanying document.
Alveolar macrophages were obtained from C 57 BL six mice and enriched as described in the video article to assess cellular activation. 125 micrograms per milliliter 50 50 CPT CPH poly anhydride nanoparticles were added to five times 10 to the fifth cells per milliliter of enriched alveolar macrophages for 48 hours. Expression of the alveolar macrophage markers, MHC two, CD 40, CD 86, and sire were then assessed by antibody staining and flow cytometry as can be seen in this histogram.
Alveolar macrophages incubated with nanoparticles indicated by the solid blue line, increased surface expression of MHC two, a molecule required for the presentation of antigen to CD four T cells as compared to those not treated with nanoparticles as expected. Lower levels of staining were seen in the fluorescence minus one FMO controls, indicating that the observed signal is not due to autofluorescence alveolar macrophages incubated with nanoparticles indicated by the solid blue line enhanced expression of CD 40. A co-stimulatory molecule necessary for appropriate activation of T cells as compared to untreated cells.
Expression of another T-cell co-stimulatory molecule CD 86 was also increased following treatment with poly anhydride nanoparticles for 48 hours. The expression of CY a C type lectin receptor that binds carbohydrate motifs present on the surface of pathogens is greatly enhanced on alveolar macrophages after poly anhy nanoparticle treatment. After watching this video, you should have a good understanding of how to perform along lavash with the purpose to harvest marine vir macrophages, how to culture those cells, and also how to stimulate those cells to measure or evaluate the cellular activation of those cells with poly hydro nanoparticles.
While attempting this procedure, it is important to use sterile laboratory techniques to prevent contamination of the primary cells being harvested and to prevent introduction of contaminants into the treatments.
