The overall goal of this procedure is to isolate primary microglial cells from the neonatal mouse cortex or the spinal cord of an adult mouse. This is accomplished by first removing the brain of a postnatal day zero or two mouse pup or an adult spinal cord. Next, either the cortices are harvested from the neonatal tissue or the spinal cord tissue is digested.
The cortical and spinal tissue cells are then cultured until experimental use. Ultimately, the harvested neonatal and adult microglial cells can be analyzed by immunofluorescent microscopy. The main advantage of our neonatal microglial culture protocol over existing methods such as shaking microglial cells for mixed cortical plates is that cells readily return to a resting state and can provide a more accurate picture of microglial cell behavior in vivo.
The main advantage of culture in microglia from the adult spinal cord is that the cells can be cited in the context of pathologies that mainly affect the adult central nervous system Prior to the dissection. Preco 10 centimeter tissue culture dishes with five micrograms per milliliter of poly de lycine or PDL diluted in autoclave water After three hours at 37 degrees Celsius, aspirate the PDL and wash the plates once with autoclave water just prior to beginning the neonatal microglia dissection. Dry the plates under the UV light in a tissue culture hood for 20 minutes.
Then use a Kim wipe, soaked in 70%ethanol to disinfect the heads of the anesthetized postnatal day zero to two pups after removing the heads with a pair of scissors. Place four heads per plate in 10 centimeter Petri dishes containing ice cold hanks buffered saline solution. Using curved forceps, anchor the heads through the eye sockets and then use straight micro forceps to carefully remove the skin covering the skull.
Then beginning near the cerebellum, remove the cranial bones, taking care not to puncture or damage the cortices. Use a small spatula to remove the brains pooling the organs in a fresh Petri dish containing ice cold HBSS. Now starting on the ventral side of the brain.
Hold the cerebellum with the micro forceps to anchor the tissue, and then make two small incisions on either side of the midbrain without cutting all the way through the tissue. Gently tease the midbrain and cerebellum in one piece from the cortices, which should form a concave of shape. Then separate the isolated cortices and orient a single cortex with the medial side up for further dissection.
In both the neonatal and the adult protocols, one of the most complex steps is adequate removal of the meninges. It is also difficult to remove the hippocampus. In the neonatal protocol, we use ice cold hanks buffered saline solution to stiffen the tissues and also use a slow, methodical dissection procedure to minimize shredding.
Remove the crescent shaped hippocampus located opposite the olfactory bulb, which appears as a small nodule on the pointed end of the cortex. Then flip the cortex over to view the dorsal side. Next, using the olfactory bulb as a starting point, remove all the meninges and the olfactory bulb itself from the cortex.
Then pull the dissected cortices into a 15 milliliter conical tube containing 14 milliliters of ice cold HBSS on ice. Now aspirate the HBSS from the Cordis and use a P 1000 pipette tip to tritrate the cortices a few times. Add four milliliters of trypsin EDTA to the tissues, and then incubate the tissues at 37 degrees Celsius.
After 15 minutes, stop the enzymatic reaction with four milliliters of complete microglial media and then spin down the cell suspensions for five minutes at 1000 times G and room temperature. After a second wash with four milliliters of complete media resus, suspend the cell pellet in 10 milliliters of complete microglial media, and then filter the cell suspension through a 40 micron mesh cell strainer. Finally plate the cells at a density of eight cortices per 10 milliliters of microglial media in one of the PD L coated 10 centimeter tissue culture plates and incubate the cells at 37 degrees Celsius and 5%carbon dioxide.
After 10 days of cell culture, detach the microglia by adding 400 microliters of 60 millimolar lidocaine in HBSS to the tissue culture plate, and incubate the plate at room temperature for 10 to 15 minutes. Then collect the resuspended cells. Rinse the plate once with HBSS and collect the wash to recover any remaining microglia.
Next, add 0.5 molar EDTA to the cell suspension to a five millimolar final concentration, and then spin down the cells resus. Suspend the palate in one milliliter of DMEM with 1%FBS and determine the number of viable cells by trian blue exclusion. Then culture the cells at the desired experimental density.
For example, for analysis by immunofluorescent microscopy plate the cells at 2.5 times 10 to the fourth cells per 18 millimeter cover slip for adult microglia tissue collection. Begin by using 70%ethanol to clean the spine of a euthanized adult mouse. Then use a pair of scissors to cut the skin above the spinal cord and proceed to cut the cord out from region T one to T 12.
Cut the remaining muscle off of the sides of the spinal cord, and then gently holding the spinal cord with the fingertips of one hand. Use micro scissors to slowly cut the vertebrae without puncturing the cord. Then use a pair of micro forceps to slowly extract the spinal cord, submerge the cord in a Petri dish containing ice cold HBSS, and then remove all the visible meninges under the microscope.
Now cut the spinal cord into transverse segments, no thicker than two millimeters to facilitate efficient digestion, and then transfer the fragments to a 15 milliliter conical tube containing one milliliter of ice cooled HBSS on ice. To digest the spinal cord tissue fragments begin by aspirating the HBS from the spinal cord tissue, being careful not to disturb the tissue. Then incubate the tissue in one milliliter of trypsin at 37 degrees Celsius.
After 30 minutes, remove the trypsin and add three milliliters of primary microglia medium containing serum to halt the enzymatic digestion. After pipetting up and down a few times to dislodge the tissue, filter the cell suspension through a 40 micron cell strainer. Then mix an aliquot of the cell suspension with an equal volume of DPI and count the cells under a fluorescent microscope to determine the total viable cell number.
Finally, plate the dissociated spinal cord tissue cells in PD L coated plates at the appropriate experimental density. Perform a full media change four hours after plating to remove the excess cell debris. In this first set of images, resting and activated microglial cells are shown 24 hours after plating the microglia exhibit, ramified or rusting morphology exposure to the priming reagent bacterial lipopolysaccharide results in changes in microglial morphology to a more amoeboid shape as the cells become activated.
The eyeball one staining a marker specific for macrophage and microglial cell surface protein and which appears in green, facilitates visualization of the cells. The brightfield image shows the overall cell populations and the DPI in blue highlights the nuclei and indicates the purity of the culture. In this representative image of cells isolated from an adult mouse spinal cord, approximately seven times 10 to the fifth viable cells were obtained.
The brightfield and fluorescence microscope settings were combined to visualize DAPI positive cells as well as the hemo cytometer grid for an accurate cell count. Microglia fully attached to PD L coated culture plates after approximately two days. In culture here, microglia isolated from the spinal cord of Mac green mice, which express GFP under the control of the microglia and macrophage promoter CSF one R are shown in the brightfield image.
Microglia as indicated by the blue arrows and astrocytes as indicated by the red arrows, can be observed Once mastered. This technique can be completed in about an hour Following this procedure. Other methods like acculturating, microglia with other cell types can be performed in order to study the effect that microglia have in other cells.
Following this procedure, you should have a good understanding of how to perform the delicate dissection necessary to culture neocortical as well as adult spinal cord cells, and employ these cells in downstream applications.
