Large-Scale Purification of Porcine or Bovine Photoreceptor Outer Segments for Phagocytosis Assays on Retinal Pigment Epithelial Cells

The overall goal of this procedure is to generate large scale stocks of photoreceptor outer segments, purified from war sign, or bovine eyes. To study one of the most important functions of retinal pigment, epithelial cells, their rhythmic daily OC acid activity. This is accomplished by first casting 25 to 60%sucrose, gradients in ultracentrifuge tubes.

Next porcine or bovine eyes are dissected and the Rey are collected and homogenized. Next, the re are filtered and loaded onto the sucrose gradient and centrifuge to separate the photoreceptor outer segments. Finally, the orange band containing the photoreceptor outer segments is either collected in either aliquot in stored as is, or labeled before Ali, quoting and storage.

Ultimately, automated fluorescent scanning, fluorescent microscopy or immuno blotting are used to study the phagocytic activity of cell lines, differentiated cells derived from stem cells or primary cultures in various conditions. The main advantage of this technique over the isolation of auto segments from mass or eyes for each experiment is that the same pool of material can be used for several experiments, thus permitting higher degree of reproducibility. Visual demonstration of this method is practical as a critical step lies in the ion casting because it conditions the range of isolated outer segments.

Demonstrating the procedure will be Celia Parino, a graduate student, and contently a technician from my laboratory After obtaining 80 fresh pig or cow eyes according to the text protocol, keep them in the dark and chilled on ice as much as possible. To avoid the photo bleaching of visual pigments and loss of orange pink color set up on the bench, the red safe light lamp absorbent pads and wipes. Prepare a 15 centimeter dish biohazard bags for waste collection and razor blades.

Once all the solutions have been prepared, according to the text protocol, pre chill them and keep them cold during all steps. Steady the gradient maker on a magnetic stir plate and insert the stir bar in the chamber closest to the exit where the 60%solution will be poured. Use a cutoff P 200 pipette tip placed at the exit of the tubing to achieve proper casting speed with 12 milliliters of each 25%and 60%sucrose solution.

And while stirring the 60%solution, cast a linear sucrose gradient in a pre chilled centrifuge tube, keeping the opening of the pipette tip at the surface of the continuously rising gradient solution. To carry out tissue collection, wear a tight fitting lab coat, gloves, sleeve protectors, and goggles under dim red light while holding an eye in one hand with the cutting surface turned away, use the edge of a razor blade to pierce the front of the eye. Then use the razor blade to cut the anterior eyeball into two halves.

Remove the lens and flip the eye cup inside out so that it can be held over the tip of one finger. Thus exposing the retina using the razor blade at an angle, gently scrape the retina, which will detach easily and appear as a pinkish layer off the tetum surface and cut at the optic nerve head. Transfer the retinas to one of two to 50 milliliter tubes containing 15 milliliters of homogenization solution on ice.

After collecting all retinas, shake the suspension vigorously by hand for two minutes. In order to disrupt the different cell layers, break the photoreceptor outer segments or POS off the rest of the photoreceptors, fragment the POS and homogenize the retina suspension. To remove large tissue fragments, use the double layer of clean gauze to filter the suspension three times into a 50 milliliter tube.

Gently press on the gauze to release remaining fragmented tissues. After each filtration, gently lay the crude retina preparation over six to 24 milliliters, 25 to 60%sucrose gradients. After balancing the tubes Ultra centrifuge at 106, 000 Gs for 50 minutes at four degrees Celsius.

When the spin is complete, aspirate most of the solution above the orange pink Band in the upper third of the gradient. Then using a P 1000 tip, collect the orange pink band into a beaker. Use approximately four to five volumes of wash one to dilute the solution and aliquot into as many 30 milliliter tubes as needed.

Centrifuge at 3000 GS for 10 minutes at four degrees Celsius and carefully discard the supernatant. Then with wash two solution reus, suspend the pellets before spinning again. Use wash three solution to wash the pellets.

A third time. To aliquot the POS. Use 10 to 20 milliliters of DMM to resuspend the cells.

Then use 490 microliters of DMM to dilute 10 microliters of cells and recalculate the yield and concentration in POS particles per milliliter. Decide on a final DMM volume and add sucrose to a final concentration of 2.5%Prepare the desired aliquots. Spin a single aliquot at 2, 300 GS for three to five minutes at room temperature.

Evaluate the pellet size and prepare various aliquot volumes to avoid freeze thawing more than once. To label the cells before Eloqua watting. Use 0.1 molar sodium carbonate buffer to resuspend one 10 milligram vial of ZI to a concentration of 2.5 milligrams per milliliter.

Rotate for one hour at room temperature in the dark and spin at maximum speed for five minutes to pellet remaining ZI solids. Using 10 milliliters of wash three solution or dmm resuspend the POS pellet and add two milliliters of the Fite solution for 80 eyes. Use aluminum foil to protect from light and rotate for at least 1.5 hours.

At room temperature. Use wash three solution to wash the cells twice as described earlier in the video, and then DM EM one to two times until almost no free fite is seen in the supernatant. Use DM EM to resus suspend POS and count an quat similar to unlabeled POS as seen here.

By keeping the eyes and the samples in the dark until after the centrifugation step, the sucrose gradient ban containing POS can be immediately recognized by its bright orange color. And if they are damaged, they will appear as much smaller pieces, a little like a dusty background. Typically, when isolated properly, POS should appear elongated, straight or bent.

When observed under a microscope, depending on the focus plane on the microscope, they can either appear dark or shiny. If not resuspended properly, they will stay in clumps. Isolated POS serve to test directly the phagocytic ability of mutant RPE cells.

In this example, POS are readily phagocytose by primary RPE cells from wild type mice, whereas cells from beta five integrin and MFG eight knockout mice tito less POS in the same amount of time. Quantification of POS phagocytosis can be done either by counting FSE labeled POS manually on microscope pictures by measuring fluorescence intensity or by flow cytometry after cell trypsin. This figure analyzes protein recruitment using immunofluorescence, colocalization assays, and protein recruitment or activation can be validated on immuno blocks after immunoprecipitation, or by checking for phosphorylation After its development.

This technique paved the way for researchers in the field of phosphate sein mediated phagocytosis to explore and characterize the molecular machinery in retinal pigment petal cells, and compare the findings to other phagocytic cells such as macrophages.