This protocol is significant because it uses biological information to facilitate the generation of a single cell suspension with a high cell viability. The main advantage of this technique is the ability to generate single cell suspensions that retain a high cell viability and an appropriate representation of the isolated cell types. Demonstrating the surgical procedure will be Hussein Atta, a fellow in my laboratory.
Demonstrating the tissue dissociation procedure will be Ali Hashemi, and the FACS analysis, Yaser Heshmati. After confirming a lack of response to pedal reflex in an adult anesthetized mouse, palpate the most prominent spinous process in the thoracic spine. Analgesia and antibiotics are administered prior to surgery.
Shave a longitudinal rectangle on the back of the mouse from the lower neck to just below the most prominent spinous process and one centimeter on each side of the midline. Disinfect the exposed skin three times with sequential 10%povidone iodine and 70%ethanol wipes in a circular fashion starting from the site of incision and working outward. After the last wipe, cover the mouse with a sterile four by four inch piece of gauze sponge with a window over the surgical field.
To induce the spinal cord injury, first make a 1.5 centimeter incision in the middle of the back and use sharp blunt dissection to separate the muscles from the spinous processes and the laminae of the T9, T10, and T11 vertebrae. Use fine scissors to sharply divide the interspinous ligaments between T9 and T10 and T10 and T11 before performing a bilateral laminectomy of the T10 spinous process. When the laminae had been completely excised, use the scissors to transect the spinal cord.
To completely cut the lateral columns, delicately sweep the tip of the fine scissors on both sides. Use a sterile cotton tip application to compress any bleeding. After achieving homeostasis, close the skin with 7-0 polyglactin910 continuous sutures and subcutaneously deliver one milliliter of saline solution to prevent postoperative dehydration, then return the animal to a fresh cage with monitoring until full recumbency.
Every 12 hours, holding the animal with one hand and massaging the lower abdomen with the other, use the index finger and thumb to locate and gently compress the distended urinary bladder to stimulate manual bladder expression until the animal is able to urinate on its own. For downstream analysis of the bladder cell types of interest, at the appropriate experimental time point and once the animal is adequately anesthetized, perform a midline laparotomy from the pelvis to the diaphragm and cut the diaphragm away from the ribs. Following the bone cartilage border parallel to the sternum, open the thorax along the ribs on both sides of the animal starting at the diaphragm and proceeding to the first rib.
After fixing the anterior thoracic wall over the animal's head, use fine scissors to cut away the pericardium and connect a 23 gauge needle to the perfusion apparatus. Insert the needle into the left ventricle and slowly advance the needle into the aorta without puncturing the tissue. When the needle is in place, begin infusing the tissue with PBS at a 15 milliliter per minute flow rate and use the tips of a pair of fine scissors to quickly make a small cut in the right atrium.
When the drainage is clear and a lightened liver color can be observed, stop the perfusion and free the bladder from the vascular pedicles and urethra, then place the bladder into microcentrifuge tube containing ice cold Tyrode's solution. To prepare a single cell suspension, when all of the bladders have been collected, tear a 1.5 milliliter microcentrifuge tube containing 100 microliters of Tyrode's solution. After weighing, mince the bladders in a 10 centimeter Petri dish containing 100 microliters of Tyrode's solution and use a wide bore pipette tip to transfer the tissue fragments into 2.5 milliliters of digestion buffer per bladder.
Incubate the tissue for 40 minutes at 37 degrees Celsius on a Nutator mixer and use a five milliliter pipette to triturate the sample for one minute before collecting the dissociated cells and tissues by centrifugation. Resuspend the pellet in one milliliter of cell detachment solution and place the cells on the Nutator mixer for an additional 10 minutes. At the end of the incubation, collect the cells with another centrifugation and resuspend the pellet in one milliliter of red cell lysis buffer.
After one minute, stop the lysis with the addition of nine milliliters of PBS and filter the cells through a 70 micrometer strainer into a 50 milliliter tube, then centrifuge the cells again and resuspend the pellet in 200 microliters of staining buffer supplemented with FC block. After a 10-minute incubation on ice, collect the cells by centrifugation and resuspend the pellet in the appropriate volume of fluorophore conjugated antibody master mix for 20 minutes on ice protected from light. At the end of the incubation, wash the cells with one milliliter of cell staining buffer and resuspend the pellet in 100 microliters of fresh cell staining buffer supplemented with five microliters of FITC Annexin V and one microliter of propidium iodide.
After 15 minutes at room temperature, add 400 microliters of Annexin binding buffer to the cells and mix by inversion. To analyze the cells by flow cytometry, first use an unstained cell sample to set the side and forward scatter parameters of the cells, then measure the fluorescence emission at 530 nanometers and greater than 575 nanometers to exclude the dead cells. Use the grids on each dot blot to define the negative population in the first decay and use the fluorescence minus one controls to correct the spectral overlap until the negative and positive population mediums are aligned.
Then measure 100, 000 events of the cells with the specific markers of interest, creating gates for the cell populations of interest as necessary. When all of the samples have been run, analyze the data using the appropriate flow cytometric analysis software. Collagen 1A1, collagen 3A1, collagen 1A2, and collagen 6A1 are the most abundant collagen types found within the mouse bladder.
The analysis to determine the mRNA expression levels of collagens one, three, six, and hyaluronan reveals that the expression of these extracellular matrix components is more prevalent in mesenchymal cell populations than in the urothelium. Flow cytometric analysis indicates that multiple different enzymatic digestion protocols yield highly viable cell populations with the third digestion protocol used in this analysis determined to be the most effective at cell preservation. Using this method, a significant increase in the total number of cells isolated from the bladders of spinal cord injury mice can be obtained to compare it to control bladders.
Compared to controls, the bladders from spinal cord injury animals also display a significant increase in CD45 positive cells. Step 7.1 is important for identifying in cardiac population. The gating strategy should be informed by the cells of interest.
The bladders from SCI animals have distinct cell population. This protocol can be used for cell sorting, primary cell culture, single-cell sequencing, and other molecule and cellular downstream procedures that require viable single cells. This protocol provides high viability and therefore low cell population loss which is a key point for single cell analysis techniques such as single cell RNA sequencing.
