This procedure begins with removing the lenses from two to four day old rats and cleaning them of any adhering tissue. The posterior side of the lens is identified and a small tear is made in the capsule. The capsule is then peeled downward and pressed into the bottom of the dish.
This process is repeated around the entire lens. The lens fiber mass is removed by rocking its sideways. The peripheral epithelium is cut away and thes are then cultured.
Finally, they are transferred to a test tube for biochemical analysis or onto a glass slide for microscopy. Hi, I'm Peggy Lanka in the laboratory of Molecular and Developmental Biology in the National Eye Institute at NIH. Hi, I'm Al Tu, also from the Laboratory of Molecular and Developmental Biology in the National Eye Institute.
I'm Chung Ga, also from same laboratory at NEI. Today we'll show you a procedure for ex planning and culturing lens epithelial from newborn rats. We'll show you how to remove the peripheral region of the epithelium, which contains cells in the early stages of differentiation.
Then we'll show you how to induce the remaining central lens epithelial cells to differentiate to form lens fibers by exposing them to FGF. We use this procedure in our laboratory to study the timing of evens involved in the terminal differentiation. We harvest the EXPLAN at various times after adding FGF to the culture medium.
Then we analyze protein expression by western blotting, RNA expression by R-T-P-C-R or micro arrays and immunostain the explan to determine the subcellular location of proteins of interest. So let's get started by moving lenses from newborn rats. To begin, use micro dissecting scissors to remove the lenses from the eyes of euthanized two to four ded rats by making a small posterior incision and pressing on the opposite side.
Transfer the lenses into a 60 millimeter plastic tissue culture dish containing five milliliters of warm sterile suspension, medium Using 0.1 millimeter tipped tweezers. Clean any adhering tissue from the lenses and transfer them to a second 60 millimeter culture dish containing five milliliters of sterile suspension medium. This helps to reduce contamination with the tweezers.
Transfer the desired number of lenses to a 35 millimeter culture dish containing two milliliters of sterile unsu supplemented medium. As many as 12 x explan can be made in the center of a dish this size. Any extra lenses can be stored in the second 60 millimeter dish from earlier and placed in a tissue culture incubator at 37 degrees Celsius up to one hour before they're used to make X explan.
Now identify the posterior side of the lens. These two I can tell are sitting with their anterior side up. I'm gonna turn it on edge so you can look down it and you'll see the cold cataract, which is a opaque area in the center.
The side farthest from the opaque center from is the posterior side. So I turn it over on the posterior side. On the posterior side, you can also see these striations, which are the remnants of this tenal vasculitis.
It's a blood vessel pattern around the periphery of the lens that also marks the posterior side.Correct. Identification is critical since this is where the capsule will be opened. Opening the lens on the anterior side will tear the epithelium.
There are many ways to identify the posterior side. First, it is rounder than the anterior side, which is slightly flattened. Second, newborn rat lenses form cold cataracts as they cool to room temperature during the dissection.
If the cold cataract has not yet completely filled the lens, the posterior side is the side farthest from the opaque region. If the opacity has filled the lens, warming the dish to 37 degrees Celsius for a few minutes will reverse it. The posterior side can be identified as the cataract reforms upon cooling.
Third vestiges of the tunica VASIs may be visible on the posterior side. Finally, the posterior suture may be visible on the posterior side. Once the posterior side has been identified, turn it upward and support the lens with the left tweezers.
Then pinch the posterior capsule with the right tweezers to produce a small fold while holding the posterior capsule with the right tweezers, grasp the capsule fold with the left tweezers and pull the two pairs of tweezers in opposite directions to make a small tear in the capsule while grasping the edge of the retracting capsule with the tweezers, pull it downward first on one side, then on the other, pressing it into the plastic of the dish with the tweezers. Repeat this several times, moving all around the equator of the lens until the capsule is firmly attached to the plate. At many points, holding the capsule in place with the left tweezers, gently rock the fiber mass with the right tweezers to break the attachment between the fiber cells and the epithelial cells at the lens equator.
Now gently pull the fiber mass away, rolling it off the capsule, which remains attached to the bottom of the dish. Repeat this process with all the lenses in the dish. Fiber masses can be discarded or frozen and saved as a source of lens proteins for other studies with microdissection of whole epithelial explan complete.
Let's look at micro dissecting and culturing central explan. Using a sterile scalpel, trim away the peripheral epithelium, which contains cells in the early stages of differentiation. Leaving a central square, approximately two millimeters on each side that consists of central epithelial cells.
Only transfer the dish containing the central x explan to a biosafety cabinet. Wash the eggplants three times with sterile PBS and once with one milliliter of fresh equilibrated suspension medium. Next, add two milliliters of culture medium to induce differentiation and place the explan in a humidified tissue culture incubator at 37 degrees Celsius, 5%carbon dioxide.
The culture period may last as little as a few hours or as long as two to three weeks depending on the parameter being studied. The medium should be changed every two to three days with microdissection and Turing complete. Let's begin harvesting the explants for analysis.
At the end of the incubation period, remove the culture medium using micro dissecting tweezers. Gently loosen the edges of each x explan. Grasp each explan with the tweezers and transfer it to a tube containing about 100 microliters of SDS lysis buffer for protein analysis or RNA later for RNA analysis, agitate the tip of the tweezers in the solution to ensure that the explan does not stick to the tweezers.
The explan are now ready for analysis of protein or RNA explan may also be analyzed by immunofluorescence. To begin rinse explan briefly in PBS. Fix the tissues by adding 4%para formaldehyde for 30 minutes.
At room temperature, remove the fixative and replace it with PBS Fixed explan become somewhat stiff, allowing them to be lifted and transferred to glass slides for immuno staining. Place a small drop of PBS without calcium and magnesium on the slide to help position the tissue and prevent curling. Using micro dissecting tweezers, lift the explan and insert it into the drop on the slide without touching the explan carefully use a paper wick to remove the liquid and flatten the explan on the glass.
Let the tissue dry in air at room temperature for three to five minutes. The implants are now ready for immunofluorescence analysis. Shown here is an example of a microdisect whole epithelial explan that has been immunostain for NCO adhere a marker for cells.
In the early stages of differentiation, only the cells in the peripheral region of the epithelium are positively stained for this protein cutting away. The peripheral epithelium as described in this protocol, leaves a central explan, which contains few, if any, cells that express and coherent. This is shown by western blotting, the central and peripheral epithelial tissues for and coherent western blotting for GA.DH was used to confirm that the protein samples were equally loaded.
The timing of events associated with differentiation can be determined by culturing central explan for different periods of time. Central explan were cultured for 2 5 24 and 48 hours in medium width or without 100 nanograms per milliliter. FGF The X explan were harvested at the indicated times and transferred to SDS sample buffer for analysis by western blotting.
The results show the end coherent protein expression is induced after 24 to 48 hours. In culture. Western blotting for GA DH demonstrates that the protein samples were equally loaded.
Immunostaining central Explan cultured for four days in the absence or presence of 100 nanograms per milliliter. FGF shows that N cadherin immunofluorescence is strongly elevated in explan that were exposed to. FGF immunofluorescence also shows the subcellular localization of n cadherin at the cell to cell boundaries.
So we've just shown you how to prepare in culture lens epithelials for studying differentiation. The main advantage of this system is that the cells differentiate synchronously. This makes it possible to determine the sequence of events.
In addition, we can add specific growth factors or inhibitors to the medium to activate or block particular signaling pathways. When doing this procedure, it's important to remember to guard against contamination by dissecting and sterile medium in a clean environment and by watching the explan thoroughly before culturing them. So that's it.
Thank you for watching. Good luck with your experiment. Bye Bye.Bye.
