The overall goal of this procedure is to improve the sensitivity of flow cytometry in order to quantify the specific immune cells in the injured spinal cord tissue via the removal of lipid my and debris using an opti prepp gradient system. This is accomplished by first dissociating the spinal cord tissue using both mechanical and enzymatic means then four opti prepp gradient Solutions are created and used to separate lipid myelin debris from cells like neurons, glia white and red blood cells. The cells isolated from the injured spinal cord tissue are then immuno labeled for specific immune cells like PMN neutrophils, ED one, macrophages, microglia, and CD three T cells.
These immuno labeled positive cells are then quantitatively assessed by flow cytometry. Ultimately, results show the removal of lipid myelin debris enhances the quantitative detection of cellular inflammation in the injured spinal cord tissue using flow cytometric analysis. The main advantage of this technique over the traditional cell dissociation method is that you can effectively separate and remove mild and debris from cells and thereby facilitate the quantitative assessment of cellular inflammation in the intraspinal cord tissue.
Using flow cytometric analysis. Begin the dissociation of the spinal cord tissue by putting Hank's buffered saline solution or HBSS in a Petri dish. Then using fine scissors mechanically dissociate spinal cord segments T eight through T 10 of uninjured control or contusion injured spinal cord tissue At T nine dissociation of an uninjured spinal cord is shown here, but the dissociation of the contusion injured spinal cord can be done in parallel.
Centrifuge the tissue bits in a 50 milliliter tube for one minute at 2000 RRP M at room temperature. Then further dissociate the spinal tissue with a trypsin and collagenase solution in five milliliters of delcos modified eagles media or DME for 20 minutes at 37 degrees Celsius before tation with a glass posterior pipette. Next, add 10 milliliters of DME plus 10%fetal bovine serum to cells to inhibit enzymatic activities, and then filter the cell solution through a 40 micrometer cell strainer into a 50 milliliter tube.
Spin the cells for three minutes at 4, 000 RPM at room temperature, then wash the pellet. Next, we suspend the cell pellet in six milliliters of HBSS for overlay on Opti Prepp gradient Solutions. Construct a diluted opti prepp solution by diluting Opti Prep one-to-one with MOPS buffer, and then make four opti PREPP gradient solutions by mixing three hundred and fifty two hundred and fifty two hundred or 150 microliters of the diluted opti prep with HBSS to a final volume of one milliliter per tube.
Now slowly and carefully, place the full one milliliter of each of the four solutions in layers in a 15 milliliter conical tube with the least dute on the bottom and the most dute on the top. Then carefully layer the six milliliters of dissociated spinal cells in HBSS, both uninjured control and contusion injured sample on top of the Opti Prep gradient solutions Next centrifuge. The tube containing the cells and gradient solutions for 15 minutes at 1900 RPM and 20 degrees Celsius.
This will separate the cell solution into distinct layers with lipid myelin debris in the top seven milliliters of the tube, followed by three layers of neurons and then inflammatory cells, glia and red blood cells in the pellet carefully aspirate the top seven milliliters that make up the lipid myelin debris layer. Then wash by transferring the remaining cell solution into a new 15 milliliter tube containing seven milliliters of HBSS. Collect the pellet by centrifugation and then resuspend the pellet in 2.5 milliliters of HBSS for immuno labeling for both uninjured control and one day post-injury sample.
500 microliters of cells are transferred into 1.5 milliliter tubes labeled separately for pm n neutrophils ED one macrophages microglia and CD three T cells cells are pelleted by centrifugation for two minutes at 5, 000 RRP M at room temperature. Resus suspend the cell pellet in 500 microliters of 0.85%ammonium chloride for five minutes to lye the red blood cells incubate for five minutes at room temperature. Then centrifuge again for two minutes at 5, 000 RPM.
Then wash the cells with 500 microliters of HBSS and centrifuge. Again, cells are blocked for non-specific antibody binding for 30 minutes with 100 microliters of 10%normal rabbit or mouse serum wash the cells. Again, then incubate the cells with fluorescent ZI antibodies against PMN neutrophils, ED one macrophages and microglia CD three T cells or isotype control IgGs diluted in HBSS for one hour.
Then wash the cells two more times and resuspend the pellet in 300 microliters of HBSS and transfer to fax tubes. Analyze the cells on a flow cytometer reading 5, 000 events per sample for all cell samples. Set flow cytometric gates using control IgG isotype labeled cells or spinal cord cells from uninjured control animals to set baseline values for normalization.
For example, set a gait for uninjured control with 1.2%cell background and detect 6.1%PMN in the spinal cord tissue. After one day post-injury removal of lipid myelin debris increases PMN neutrophil detection in the injured spinal cord one day post-injury or DPI as shown in this figure, PMN neutrophils quickly enter the spinal cord starting as early as two hours post-injury and peak at one day post-injury. Here it can be seen that ED one macrophage microglial numbers increase in the injured spinal cord starting at three days post-injury peak acutely at seven days post-injury drop to low levels at 14 days post-injury before rising to a surprising second peak at 60 days post-injury, and remain in the injured spinal cord for up to 180 days post-injury.
In this figure, the time dependent multiphasic response of cellular inflammation after spinal cord injury is shown. The initial phase of cellular inflammation is composed of the early peak of PMN neutrophils at one day post injury, followed by a peak of ED one macrophages, microglia at seven days post-injury and CD three T cells at nine days post-injury. The later phases are composed of all three cellular populations, which rise in number after 14 days post-injury, and persist through 180 days post-injury with a notable second peak of macrophages, microglia at 60 days post-injury injury.
This method may be useful to neuroinflammation research and may facilitate the investigation of specific immune cells in traumatic brain and spinal cord injuries, as well as other pathological condition in the central nervous system.
