The overall goal of this procedure is to visualize nano podia through immunofluorescent staining in cultured cells. In vitro. This is accomplished by first culturing the cells of interest on collagen coated glass discs.
In the second step, the cells are fixed to the disc with warm PFA next, the cells are washed and then incubated in blocking buffer for one hour at room temperature. In the final step, the cells are stained with antibody against TM four, SF one, and mounted on a glass slide. Ultimately, the presence of nano podia extensions in mobile proliferating or interacting cells can be visualized by immunofluorescence microscopy.
The implications of this technique extend toward the therapy and a diagnosis of human cancers because endot cell and tumor cell proliferation movement and intercellular interactions are key processes involved in the angiogenesis and growth of tumor cells. Demonstrating the procedure will be two lean, a postdoc from a laboratory Begin by autoclaving 12 millimeter glass discs in a four ounce glass jar while the discs are being sterilized. Place a 50 milliliter conical tube containing 25 milliliters of 70%ethanol into a cell culture hood.
Place a sharp forceps with an extra fine point into the ethanol for five minutes, and then carefully remove the forceps and cap the tube, placing the forceps on top of the tube to air dry for two minutes. When the forceps are dry, place one sterile glass disc per well into one of each of the desired wells of a 24 well cell culture plate. Then cover each disc with 500 microliters of bovine collagen solution and place the plate in a 37 degree Celsius 5%carbon dioxide cell culture incubator for at least 30 minutes.
While the discs are incubating drip and eyes the cells of interest stopping the reaction with complete culture media. When the cells have detached, collect the floating cells in a 15 milliliter Falcon tube and then count them after confirming that their viability is greater than 90%Pellet the cells for 10 minutes at 200 G and four degrees Celsius. Remove the supernatant and then dilute the cells to one times 10 to the fifth cells per milliliter in cell culture media, and place the cells on ice.
Now aspirate the collagen solution from each well in the cell culture hood, and then gently dispense 500 microliters of the cells onto each collagen coated disc culture. The cells for the appropriate amount of time to track the nano podia activities and cell passage. Typically, cells will attach to the glass disc in the first 30 minutes, start to polarize and migrate in the first hour, and perform intercellular interactions and cell division in the following hours to fix the cells first warm enough PFA for all the discs in a 37 degrees Celsius incubator for 30 minutes.
Then place a heating pad in the culture hood. Turn it on, and then place the prewarm PFA on top of the heating pad. Adding PFA to the discs is the most crucial step for preserving the original cellular structure of the cell activities to facilitate the removal of the culture medium and the addition of the PFA without disturbing the cells in culture, tail the plate to about 45 degrees, leaning it against a styrofoam stand so that it is stable at the angle Using one hand.
Next, aspirate the medium from each well then immediately use the other hand to slide 500 microliters of PFA into each well through the well edge and incubate the discs for another five minutes. Next, remove the PFA as just demonstrated for the cell culture media dispensing the used PFA into a collection tube with one hand and immediately replacing the PFA with 500 microliters of room temperature PBS with the other after the wells have been washed twice with PBS, remove the final PBS wash as just demonstrated and add 500 microliters of immunochemistry blocking buffer to each disc. To visualize the nano podia by fluorescence microscopy, first replace the immuno cyto chemistry blocking buffer in each well with 0.01%tritton X 100 blocking buffer at room temperature for one hour.
Then replace the tritton blocking buffer with 300 microliters of freshly diluted anti TM four SF one antibody solution for two hours at room temperature. Next, wash each disc three times with PBS as just demonstrated, leaving the PBS on the discs for five minutes during each wash after the last wash. Incubate each well with 300 microliters of freshly diluted secondary antibody and Phin solution and incubate the discs for another two hours at room temperature.
Now wash the discs three times again, leaving the final PBS wash in the wells for an hour. Then at a single 10 microliter drop of Antifa mounting media onto one glass slide for each disc. Next, gently bend the tip of a 19 gauge, one and a half inch syringe needle 90 degrees on a hard surface.
Then holding the needle in one hand, gently lift the glass disc one by one using sharp forceps to grasp the disc with the other hand. Place each glass disc face down onto the mounting media on the glass slides, and then carefully place the slides in a dark place at room temperature to dry overnight. The stained nano podia can then be imaged or stored in a slide box at minus 20 degrees celsius.
Normally growing cells will attach to the collagen coated discs within 30 minutes after seating polarize and become mobile soon after extending nano podia ahead of their path of movement. As demonstrated in these images, isolated individual H-U-V-E-C generally will show a clearly polarized morphology with f actin projections at the cell periphery embedded inside the nano podia PC three cells will display a polarized morphology with their nano podia revealing the path of PC three cell movement. If the cells are in the state of cell division, as seen here in a dividing H-U-V-E-C, the nano podia firmly attached to the matrix through TM E and permit F actin to extend or retract inside the nano podia.
Intercellular interactions leading to cell junction formation are also commonly observed through nano podia suboptimal temperatures as shown in these examples using four degrees Celsius PFA fixation and cold PBS washing will disturb and destroy the nano podia structure in polarized cells. In cold PFA and cold PBS proliferating cells will lack podia and thus f actin retraction within the nano podia will be observed. And gaps at the intercellular junctions will be artificially produced as nano podia are thin long membrane projections that attach to the extracellular matrix through teamed.
The teamed intensity will likely need to be enhanced through the brightness and contrast functions in image processing software to reveal the path of cell movement through nano podia residues. As was done in the inset of this image here, H-U-V-E-C that were cultured for six hours after plating at a 60%co fluency on a collagen coated glass disc, and then fixed in 37 degrees Celsius, 4%PFA as just demonstrated are shown in this image. The nano podia were revealed with anti CD nine antibody and F actin was stained with Phin demonstrating that CD nine also localizes to the nano podia.
Following the procedure. Other methods like flow cytometry in western blood can be performed to answer additional questions like, how do certain treatments influence TM four SF one expression levels and thus affect cell movement proliferation and intracellular interactions. After watching this video, you should have a good understanding of how to perform immuno frozen sustaining for studying the role of nano podia in cell movement and intercellular interactions.
