The primary goal of this procedure is to monitor prion uptake and trafficking by immune cells within hours after infection. To start purify and fluorochrome label aggregated prion rods from infected brain, then inoculate these prion rods into the peritoneal cavity of mice After two hours, harvest cells from the peritoneum spleen and also the mediastinal and mesenteric lymph nodes. Finally stain recovered cells with immune phenotypic markers and analyze them for PreOn retention.
For instance, the flow cytometry results can be used to show that prion rods are retained and trafficked primarily by macrophages and dendritic cells, secondarily by monocytes and tertiary by B cells. This experimental system is designed to shed light on prion reservoirs and potential therapeutic targets by answering key questions such as what cells take up PreOn immediately after infection. Where do they take them and how fast do they get there?
Demonstrating this procedure will be Crystal, Mt.And Brady Michael, graduate students from my laboratory In order to collect the PreOn proteins. Homogenize 100 milligrams of PreOn infected brain tissue in 900 milliliters of ice cold homogenizing buffer for one minute at maximum speed in a commercial blender immediately place on ice for two minutes. Repeat this process three times to increase protein yield.
Centrifuge the homogenate for 10 minutes at 3000 Gs and four degrees Celsius. Remove and save the snat on ice. Resuspend the pellet in one liter of hp and repeat the homogenization and centrifugation steps.
Now, pool the snat and centrifuge for 60 minutes at 100, 000 GS zero degrees Celsius. Discard the supernatant. Resuspend the pellet in 100 milliliters of TBST.
Estimate protein concentration by the Bradford Assay and adjust volume to five milligrams per milliliter protein in TBST. Chill on ice for 30 minutes. Again, centrifuge for 30 minutes at 100, 000 GS zero degrees Celsius.
Discard the SNA and wash the pellet first with 50 milliliters of TBST. Then 100 milliliters of TBS re suspend the resulting pellet in 100 milliliters of one XPBS containing 1%sarcas seal and proteus inhibitor cocktail and stir for 120 minutes at 37 degrees Celsius. To perform a sucrose gradient, layer the sample on 900 milliliters of 320 millimolar sucrose and centrifuge for 60 minutes.
At 100, 000 GS 10 degrees Celsius. Discard the snat and re suspend the pellet in 10 milliliters of 2.3 molar sodium chloride, 5%Sarco seal. Now sonicate the sample five times 40 seconds at 70%maximum power at one minute intervals.
Eloqua one milliliter of sample into einor tubes and centrifuge for 15 minutes at 13, 000 GS four degrees Celsius. Wash pellets twice with TBS and store stocks at minus 70 degrees Celsius. Conjugate one tube of purified PreOn rods using one vial of DITE 6 49 fluorochrome according to the manufacturer's protocol.
Finally, exchange the dite labeling buffer for PBS by centrifugation through filter columns. Store in 20 microliter aliquots at minus 70 degrees Celsius away from light until ready to use. This section encompasses intraperitoneal, prion, inoculation, and recovery of cells from the peritoneum, mediastinal, and mesenteric lymph nodes and spleen immediately prior to use D loop fluorescent prion.
Rods one to 50 in PBS scruff mice by the dorsal neck fur with thumb and index finger. Gently turn the mouse to face you and restrain the tail with your pinky finger. Supinate the mouse, elevating the hind torso above the head and holding him slightly upside down.
Using a 30 gauge insulin syringe, inject 100 microliters of fluorescent pion rods, one centimeter lateral of the midline and one to two centimeters anterior to the scro iliac joint. Insert the needle one centimeter through the skin directed 45 degrees medially for control mice, use a one to 50 dilution of fluorescent beads or PBS alone. As per experimental design, incubate the mice for the allotted time to begin analyses.
Euthanize the mice according to approved animal care and use committee protocols. Inject 10 milliliters of PBS into the peritoneal cavity using a 12 milliliter syringe and 25 gauge needle. Gently rock the mouse longitudinally for one minute to continue with the mouse dissection.
Wet the ventral side of the mouse coat thoroughly with 70%ethanol. Lift the skin at the midline just below the rib cage with forceps and cut a small incision through the skin with scissors. Make a three to five centimeter long incision through the skin in both directions to head and tail than two.
Two centimeter incisions from the midline laterally from each end, peel back and pin the skin to reveal the peritoneal and thoracic cavities. Recover cell suspension from the peritoneum using the same syringe and a 16 gauge needle. Place exudate into a 15 milliliter conical tube and reserve on ice.
Carefully cut through the thin translucent membrane surrounding the peritoneum and reflect the thorax to reveal the mediastinum. Now find and remove two to four mediastinal lymph nodes, slightly dorsal and laterally adjacent to the thymus medially adjacent to the brachiocephalic artery near the ventral side of the trachea. Next, find and remove up to six mesenteric lymph nodes embedded in the soft white mesenteric tissue that anchors the intestines to the posterior abdominal wall.
Reserve lymph nodes in one milliliter of PMI 1640, medium on ice until the section is complete. Finally, find and remove the spleen. A long dark red organ located just lateral of the midline and dorsal to the intestines on the left side of the mouse.
Using the plunger of a one milliliter syringe macerate and press lymph nodes through a 40 micron mesh strainer into three milliliters of PBS in a plastic petri dish. Rinse the strainer with an additional two milliliters of PBS and transfer the total five milliliter cell suspension to a 15 milliliter conical tube. Also, rinse the Petri dish with an additional five milliliters of PBS and transfer to the same tube.
Centrifuge the sample for five minutes at 250 gs. Discard the supernatant and resuspend the cell pellet. In one milliliter of fax buffer transfer isolated cells to a 1.5 milliliter EOR tube.
Typically, this staining protocol can be used for identifying immune cell subsets, using fluorescent antibodies against well-characterized immune cell surface markers to lu all antibodies into fax buffer. Immediately before use. Count the cells and aliquot 10 to the sixth cells into einor tubes, pellet cells by centrifugation and wash the pellet two times with one milliliter of fax buffer to block endogenous FC receptors.
Incubate the cells in 100 microliters of two nanograms per milliliter rat anti-US CD 1632 for 20 minutes on ice pellet and wash cells with fax buffer. Next, add 100 microliters of one nanogram per milliliter of appropriate fluorescent antibody or antibodies to each sample and incubate on ice. For one hour, add 100 microliters of fax buffer alone to control cells, pelle and wash cells twice with fax buffer.
Re suspend the cells in one milliliter of act buffer, incubate on ice for one minute. To lyse red blood cells pellet and wash cells with fax buffer resuspend cells in one milliliter of fax buffer. Analyze the samples on a flow cytometer typically with four hundred and five, four hundred and eighty eight and 633 nanometer excitation lasers, and five to 10 fluorescent emission detectors.
Crude prion, infected brain homogenate is greatly enriched for prions. The purification process involves detergent, solubilization, and ultracentrifugation of the E two elk brain homogenate. The product is protease.
K resistant fax analysis of specific cell populations demonstrates that antigen presenting cells home to the peritoneum by two hours and retain the fluorescent beads and prion rods. Cells from bead and prion. Inoculated mice displayed significant fluorescence, especially on monocytes, dendritic cells and macrophages.
PreOn bearing monocytes, dendritic cells, and macrophages were also found in the mediastinal lymph nodes. Two hours after inoculation, we detected no beads or PreOn rods in the or mesenteric lymph nodes. In this experiment, the video demonstrated several protocols to purify aggregated prion rods from infected brain to perform intraperitoneal injections, to correctly identify and isolate lymph nodes, and finally to isolate and stain immune cells.
