Immune cells infiltrate the central nervous system during infection, trauma, autoimmunity, or neurodegeneration. While histochemical approaches can be used to determine the location of the infiltrating cells and to analyze the associated pathology, these techniques are limited by the number of antibodies that can be used simultaneously. Here a method for rapid isolation of mononuclear cells from brain and spinal cord tissues is shown in which the dissected tissue is first homogenized.
Next, a perol gradient is set up and following centrifugation, the mononuclear cells are collected from the interface. Finally, the cells are rinsed and stained with antibodies Through flow cytometry analysis, hematogenous cells and surveillance microglia are distinguished and their relative proportions during disease states can be determined. The main advantage of this technique over existing methods like Aris gradient or in such a histochemistry, is that it is less time consuming.
This allows you to process more tissues at the same time. Demonstrating the gradient setup will be palino A technician from my lab Prepare the reagents that will be needed for this protocol. Starting with four milliliters of stock isotonic perol referred to as SIP per brain.
To do this, combine nine parts of perol with one part 10 times HBSS without calcium and magnesium mixed by inversion. Next, prepare 70%per call while mixing the prepared SIP with one times HBSS without calcium and magnesium. Then dispense two milliliters of the 70%per cole into a 15 milliliter polypropylene conical tube to collect tissue.
For this procedure, place the dissected brains and spinal cords of all the study mice in beakers containing 10 milliliters of RPMI over ice. Next, transfer the tissue to a seven or 15 milliliter down homogenizer containing three milliliters of one times HBSS with a loose fitting size. A pestle gently make a cell suspension, then switch to a tight fitting size B pestle, and further dissociate the tissues.
Once the tissue is homogenized, add RPMI or one times HBSS to a final volume of seven milliliters and proceed to setting up the gradient to set up the percoco gradient. Begin by adding three milliliters of SIP to the cell suspension to a final PERCOCO concentration of 30%Keep in mind that the perol should be used at room temperature. If it is used cold, the cells tend to clump and cell separation is less efficient.
Next, using our pipette aid setting gravity mode, slowly layer the 10 milliliter cell suspension on top of the 70%SIP. Avoid mixing the 70%and 30%solutions. This is the most critical step of the procedure.
A very clear flat line should be visible at the 70 to 30%junction. Generally, individuals new to this method will struggle because the third and 70%solutions will mix during the layering. Next step, compromising the interface Centrifuge 30 minutes of 500 Gs at 18 degrees Celsius.
Make sure the centrifuge will stop with minimal or no break so that the interface is not disturbed. After spinning the gradients, a thick and viscous layer of debris should accumulate at the top of the tube. The next layer is the yellowish 30%per core layer, which sometimes contains debris.
Next is the 70 to 30 interface, which should have a defined white halo and contains the mononuclear cells. Finally, at the bottom of the tube is the clear 70%perol layer. If no interface is observed, the yield of cells will likely be very low, making further analysis extremely difficult.
Therefore, at that point, fresh cell suspensions might be required to decrease contamination. Carefully remove the layer of debris from the top of the tube and discard it. Then using a plastic pipette, go through the top 30%per core layer and collect two to three milliliters of the 70 30 interphase and transfer it to a clean conical tube containing eight milliliters of one times HBSS.
Mix the diluted interfaces a few times by inversion, then centrifuge for seven minutes at 500 GS at 18 degrees Celsius. Following the spin, remove the supena and resuspend the pellet. In one milliliter of cell staining buffer, transfer the cell suspension to a 1.5 milliliter tube and wash it one more time.
Using a micro centrifuge at 10, 000 GS for one minute at four degrees Celsius before proceeding with antibody staining to perform antibody staining, resuspend the pellets in 50 microliters of purified anti-US CD 16, CD 32, diluted one to 200 in cell staining buffer to block the FC binding sites. As for any staining procedure, it is imperative that all antibodies are adequately titrated incubate on ice for 10 minutes. During the incubation, remove two to five microliters of cells and count them using a hemo cytometer.
The combined naive brain and spinal cord tissues should yield around three to five times 10 to the five cells per mouse. Inflamed brains will have ranging numbers of inflammatory cells depending on the time point at which they are collected and the disease model Following the incubation with CD 16 and CD 32, add 50 microliters of immune cell specific antibody cocktail mix. The mix used in this example contains antibodies against CD 45, CD four, and CD 19 incubate on ice for 30 minutes.
Importantly, each antibody must be first titrated. To assess optimal dilution, use appropriate fluorochrome combinations chosen according to the capabilities of laser and filter settings of your particular flow. Cytometer, after staining the cells, wash them in cell staining buffer by adding one milliliter of cell staining buffer and spinning them for one minute.
At 10, 000 Gs following the spin, discard the supine agent, then resuspend pallets in 100 to 200 microliters of cell staining buffer for immediate flow cytometric analysis for later analysis. Reus suspend the cells in 2%par formaldehyde prepared in PBS and store overnight at four degrees Celsius. Shown here are mononuclear cells obtained and incubated with FC block, followed by anti CD 45, CD four, and CD 19 antibodies.
In accordance with the demonstrated protocol by flow cytometry CD 45 staining nicely separates CD 45 high hematogenous cells from CD 45, DIM surveillance microglia. This figure compares the cells isolated from naive and an inflamed brain from a mouse induced to develop experimental autoimmune encephalomyelitis. CD 45 A PC intensity is shown on the Y axis where three distinct populations are visualized in the naive brain.
Very few numbers of CD 45 high cells are detected. Usually less than 2%of the total isolated population. In comparison brains from EAE affected mice show a massive influx of blood cells recruited to the brain.
Addition of other cell surface markers such as CD 11 b, CD four, CD eight, et cetera, allows further characterization of the CD 45 high population Once mastered, this technique can be done in three to four hours if it is performed properly. While attending this procedure, it is important to remember that histological approaches in conjunction with immune phenotyping by flow cytometry are critical to fully characterize the composition of the CNS inflammatory infiltrates.
