The overall goal of this procedure is to isolate cortical microglia from murine neonates. This is accomplished by first micros dissecting cortical brain tissue from Muren neonates. The second step is to grow mixed glial cell cultures for 17 to 21 days in vitro.
Next, the microglia are isolated from these mixed glial cultures. The final step is to plate microglial cells in cell culture plates for further experimentation. Ultimately, immunofluorescence microscopy and Eliza are used to show that this isolation protocol preserves microglial immuno phenotype and functionality.
Visual demonstration of this method is critical as the SEP involved in micro microdissection are difficult to perform. Successful execution of this procedure requires both speed and accuracy. That is being too slow will reduce your cell yield while incomplete removal of the meninges will contaminate the glial culture and impede glial growth.
Begin by preparing the microglia complete media referred to as MCM hereafter. Prepare one flask per pup and incubate them for at least one hour. This equilibration step is essential for success.
Next, prepare aliquot and chill on ice. All required media aliquot six milliliters of MCM for each pup and warm in a 37 degree Celsius water bath prior to dissection. Wipe down the horizontal airflow workstation and dissection microscope with 70%ethanol.
Next place the sterilized surgical tools needed for the procedure into the work area. Prepare a sterile dish with dissection media for capturing the head and another for dissection. Dispense the mincing media into its own dish for mincing the brain tissue.
After decapitation and brain extraction, place the freshly isolated brain under a dissecting microscope to visualize the courtesies, making sure to keep the dorsal side up and dry. Secure the brain by inserting the tips of forceps into the intersection of the midbrain and cortex. Once in place, completely remove the dorsal cortical meninges.
Next, remove the ventral meninges to maximize microglia purity and yield. It is essential to completely remove all meningeal tissue. Separate the cortices from the remainder of the brain.
Be sure to remove any residual cortical meninges that may be present on the ventral or medial side of the cortices. With the meningeal tissue removed, place the cortices into the mincing dish prepared earlier. Working in a class two biological safety cabinet, use scissors to mince the brain tissue.
Transfer the minced tissue into a 15 millimeter conical tube. Rinse both the scissors and mincing dish with one milliliter of mincing media each to maximize yields. Collect and dispense these rinses into the 15 milliliter conical tube containing the minced tissue.
Leave the tissue suspension undisturbed for one to two minutes, allowing the minced tissue to settle at the bottom of the tube. After this time, carefully remove and discard two milliliters of the supernatant, taking care not to disrupt the settled minced tissue. Add three milliliters of the previously warmed MCM to the minced tissue, and then tri the tissue forcefully.
No discernible solid tissue should remain when trier is complete. Add another three milliliters of MCM using the same pipette tip to collect residual triturated sample from the tip. Once collected, centrifuge the minced tissue to pellet the cells.
When complete return the pelleted cells to the biological safety cabinet. Carefully remove and discard the supernatant. Gently resus.
Suspend the cell pellet with two milliliters of warm MCM, and then add the cell resus suspension to the flask prepared earlier. Incubate the cells for 24 hours. After 24 hours, gently tap the flask against the palm of the hand to disrupt the tissue debris.
Check the cells before and after tapping to ensure the debris is completely removed. Next, replace the media with fresh MCM and return the flask to the incubator. Check the cells daily to monitor growth and contamination.
After approximately 17 to 21 days, microglial cells will be ready for harvest to determine if the cells are ready, examine them under an inverted phase contrast microscope. Microglia are ready for harvest. When they have reached approximately 40%co fluency, they appear as small, bright spherical cells growing as a monolayer on top of other.
To isolate, microglia vigorously tap the flask against the laboratory bench. This agitation helps separate microglia from other glial cells due to the low adherence properties of microglia. Seal the flask caps with paraform and rotate the mixed glial cultures using a temperature controlled non humidified rotary shaker for five hours.
After this time, visually inspect that the microglia have lifted off the flask surface. Using an inverted phase contrast microscope microglia are bright spherical free-floating cells. There will be a residual layer of astrocytes and oligodendrocytes adhering to the bottom of the flask.
Next, collect the supernatant containing the microglia into a sterile conical centrifuge tube and palate.Resus. Suspend the microglial pellet in warm microglial growth medium and determine the cell concentration using a hemo cytometer plate. The cells on a 24 well plate format on plastic or sterile glass cover slips 24 hours later.
Replace the media with fresh warm MGM to remove floating cells and debris. This step is critical to cell survival and to maintain quiescent microglia, microglia cells are ready for experimentation immediately after this refeeding step to assess microglial purity. Cell cultures derived from microglia endogenously expressing GFP were stained for the macrophage antigen IBA one and counter stained with dapi.
In order to visualize the cell nucleus, greater than 95%of the cells exhibited colocalization of GFP IBA one and dpi. After challenging the cells with LPS, the microglia adopted a stark morphological change from a small bi phenotype to an amoeboid like shape. Immuno phytochemical staining for P 65 evidence that under normal conditions P 65 is diffusely localized throughout the cytoplasm.
Whereas after a two hour exposure to Pam P 65 immunoreactivity dramatically shifted localization to the cell nucleus to confirm that isolated microglia retained their biochemical immuno functionality. The secretion of the pro-inflammatory cytokine TNF alpha was determined after exposure to PAM or LPS. After watching this video, you should have a good understanding of how to isolate cortical microglia from mirroring neonates for the best results.
It is important to maintain sterile technique and to thoroughly remove meningeal tissue from the dissected cortical region.
