密度梯度离心法分离小鼠原发胶质细胞的研究

The overall goal of this protocol is to isolate a high-purity population of microglia from rodents. This method can help answer key questions in the neuroinformation field such identifying the immunomodulatory properties of stem cells on microglia or performing drug screening assays. The main advantage of this technique is the isolation of a high-purity population of microglia without the need for prolonged time in culture.

To begin this procedure, insert the tips of the scissors through the opening in the spinal canal and make lateral incisions to the ear canal. Then, gently slide the tissue towards the rostrum to expose the skull. Next, make an incision along the sagittal suture towards the bregma.

Then, insert the tip of the scissors into the bregma and make lateral incisions. Afterward, gently peel away the skull to expose the brain. Using curved forceps, remove it and transfer to a five-milliliter sterile container.

Wash it two to three times with five milliliters of ice-cold wash media per wash to remove the blood. In a laminar airflow hood, place the contents of the five-milliliter container in a small Petri dish resting on ice. Using a sterile scalpel blade or sterile scissors, remove the cerebellum and olfactory bulb.

Then, make a midline cut to separate the two hemispheres. Subsequently, identify the meningeal layer as a very thin layer of cells with a red tinge on the surface of the brain with visible blood vessels. Carefully peel away the meningeal layer with fine forceps taking care not to damage the cortices.

If the meningeal layer breaks, continue peeling away the torn fragments until it is completely removed. Then, transfer the hemispheres to a new small Petri dish on ice and fill it with wash media. Chop the brain into small pieces with sterile scalpel blade.

Following that, add 100 microliters of papain and 150 microliters of DNase1 into the media and incubate for 30 minutes at 37 degrees Celsius. After digestion, triturate the tissue using a P1000 pipette. If the tissue pieces are too large to enter the pipette, consider using a pair of sterile scissors to widen the pipette tip.

During this process, take care not to introduce bubbles into the media as this may reduce cell viability. In a laminar flow hood, prepare a 50-milliliter conical tube with a 100-micrometer cell strainer. Pour the digest medium and brain pieces onto the strainer.

Push through the pieces of brain by using the plunger of a sterile three-milliliter syringe in a grinding motion until there is no more tissue visible. Continuously wash the filter with the wash media throughout this process. This will wash through any cells trapped in the strainer.

After that, centrifuge the single-cell suspension at 500 G for five minutes at four degrees Celsius. Then, prepare the stock isotonic Percoll by adding nine-to-one ratio of density gradient medium to 10X sterile HBSS. Prepare gradients as 30%SIP in DMEM and 70%SIP in one-X HBSS.

For example, to prepare 10 milliliters of 30%SIP, add three milliliters of SIP to seven milliliters of DMEM. Next, aspirate the supernatant from the conical tube and re-suspend the cell pellet with eight milliliters of 30%SIP in DMEM. Transfer the full volume to a fresh 15-milliliter conical tube.

Afterward, underlay the 70%SIP solution by filling a transfer pipette with 70%SIP and carefully push through the transfer pipette to the bottom of the conical tube. Once the tip is close to the bottom, gently push the contents through the transfer pipette. Subsequently, centrifuge the SIP layers including the cells at 650 G with brake zero and acceleration four for 25 minutes at room temperature.

The 70%Percoll layer must be underlaid with great care. Disruption of this interface can severely impact the trapping of the microglia in the cell layer and severely reduce yields. Then, aspirate four milliliters of the media with cellular debris from the top of the tube to ease mononuclear cell removal in the following step.

Next, carefully lower the pipette tip towards the interface and isolate the mononuclear cells from the 30/70 density gradient medium interface. Collect approximately three milliliters from the cloudy interface and transfer it to a new 15-milliliter conical tube. Following this, dilute the mixture with nine milliliters of HBSS to aid removal of the density gradient medium.

Centrifuge the diluted density gradient medium interface containing the mononuclear cells at 500 G for five minutes. Aspirate the supernatant and re-suspend it with one milliliter of growth medium. Subsequently, stain the re-suspended cells with Trypan blue and perform a cell count using a hemocytometer.

In this step, centrifuge the collected mononuclear cells at 300 G for 10 minutes at four degrees Celsius to remove the growth medium as this will interfere with the magnetic isolation. Using the same cell count obtained earlier, re-suspend at one times 10 to the eight nucleated cells per milliliter in PBS containing 2%FBS and one millimolar EDTA within a volume range of 0.1 to 2.5 milliliters. Next, add the full volume of nucleated cells in PBS to a fresh five-milliliter polystyrene round-bottom tube.

Then, add 50 microliters of CD11b PE labeling reagent per one milliliter of sample. Incubate it at room temperature for 15 minutes protected from light. After that, add 70 microliters of selection cocktail per one milliliter of sample.

Incubate it at room temperature for 15 minutes protected from light. Subsequently, mix the magnetic particles by pipetting up and down more than five times. Add 50 microliters per milliliter of to the sample and incubate at room temperature for 10 minutes protected from light.

If the total volume of cell mixture is less than 2.5 milliliters, top up to this volume with PBS containing 2%FBS and one millimolar EDTA and mix by gently pipetting two to three times. Then, place the tube into the magnet and incubate at room temperature for five minutes. In one continuous motion, fully invert the magnet containing the tube for two to three seconds to pour off the supernatant.

Return the magnet to an upright position and remove the tube from the magnet. Following that, wash the remaining cells out from the column by repeating the procedures. Re-suspend the cells in a desired growth medium.

Rinse the side of the collection vessel to collect cells from the sides of the tube and maximize the yields. As can be seen in this figure, the isolated primary microglia retain their spherical cell body and distinct ramified structure. Here are the representative facts plots of the isolated microglia.

The combined staining of annexin V and propidium iodide allows the categorization of apoptotic, necrotic or live cells after stimulation with LPS. hAEC-conditioned medium significantly reduced microglia apoptosis relative to controls suggesting a form of protection on this cell type. Once mastered, this technique can be performed in six to eight hours if performed correctly.

When performing this method, it's important to take great care in layering the Percoll as this step will have the greatest impact on yields. Following this technique, other procedures such as the phagocytic function assay, or co-culture with neurons, can be used to answer additional questions about the immunomodulatory properties of your cell type of choice on primary microglia. After its development, this technique paved the way for researchers in the field to discover how primary microglia can be modulated in perinatal brain injury.

The implications of this technique extend toward the therapy and diagnosis of perinatal brain injury as it allows the characterization of a primary immune cell type known to be involved in neuroinformation that leads to motor and cognitive dysfunction. Though this method can provide insight into motor disorders such as cerebral palsy, it can also be applied into other disorders where microglia plays a role in the pathogenesis such as Alzheimer's disease. We first had the idea for this method when we wanted a technique for the quick characterization of microglia circumventing potential epigenetic changes after prolonged time in culture.

After watching this video, you should have a good understanding of how to isolate high-purity population of microglia for downstream characterization.