The overall goal of this procedure is to prepare primary microglial cell cultures from neonatal rat brains. This is accomplished by first isolating the brain and removing the meninges. The second step is to homogenize the brain and to plate to mixed glial culture into T 75 flasks.
Next, the flasks are incubated for 10 to 14 days until an astrocyte monolayer has reached confluence. The final step is to shake the flasks to release microglia that are growing on top of the astrocyte monolayer, resulting in a purified culture of microglia. Ultimately, high purity primary microglia can be used in subsequent in vitro single cell culture and co-culture assays.
The main advantage of this technique over existing methods like the Perico gradient method, is that nominal mechanical disruption minimizes microglial dysfunction or activation, and microglia for experiments can be generated for weeks after the initial preparation. I will be demonstrating this procedure along with Tammy Tero, a graduate student in Dr.Clifton Dau guard's laboratory. The high purity microglia cultures attained during this protocol can be used in in vitro experiments to study microglia function under normal physiological conditions, as well as pathological disease conditions.
Microglia, responsiveness to tissue damage, as well as inflammatory stimuli has direct relevance to neurological injury and neurodegenerative disease conditions. Generally, individuals new to this method may struggle because they're unfamiliar with how to remove the meninges from brain tissue in a timely manner in order to reduce fibroblast contamination in the primary microglia cultures. In addition, care should be taken not to damage the astrocyte monolayer during the shaking of flasks and handling of the microglia cultures.
To prepare for the procedure, Chi Livi is L 15 media to four degrees Celsius, ready for 60 by 15 millimeter Petri dishes containing L 15 media and place on ice. Warm the culture media to 37 degrees Celsius and ensure that all surgical tools have been sterilized. After decapitating a P one to P five rat pup with sharp scissors, drop the head into 70%ethanol.
After the heads of five rat pups have been collected, transfer the heads to saline solution. Remove the whole brain from each head by carefully making incisions on both sides of the skull above the ears, and pulling the brain out and into one of the Petri issues containing L 15 medium on ice. Once the first five brains have been transferred to L 15 on ice, the next five pups can be processed in the same manner when all of the brains have been collected.
Remove the meninges by gently grasping an edge of the meningeal sheet with forceps and carefully peeling it away from the underlying cortex. It is essential to thoroughly remove the meninges and to do so as quickly as possible. After removing the meninges, transfer each of the brains into a fresh Petri dish of L 15 medium on ice.
Use a 10 milliliter pipette to aspirate the brain tissue and medium from the plate into a sterile 50 milliliter conical tube. Then centrifuge at 2, 500 RCF for five minutes at four degrees Celsius. Following centrifugation aspirate.
The supinate then uses sterile 10 milliliter pipette to Reese. Suspend the pellet in four to five milliliters of fresh L 15 media, pipette the media and tissue up and down 10 times. Next place a 100 micron pore cell strainer onto a fresh 50 milliliter conical tube.
Use a sterile five milliliter pipette to pipette the tissue suspension up and down, and then with the pipette flushed to the cell strainer, dispense the material through the cell strainer into the conical tube. Rinse the strainer with four to five milliliters of fresh chilled L 15 media. Then centrifuge your 2, 500 RCF for five minutes of four degrees Celsius.
For each rat pup brain processed, prepare a sterile T 75 flask by adding 12 milliliters of pre-war culture media into each flask. Next, after aspirating the sup natant from the pelleted cells, add five to six milliliters of culture media to the cell pellet, pipette up and down 10 times with a 10 milliliter pipette. Then transfer an equal volume of cell suspension to each T 75 flask.
Incubate the flasks in a 5%carbon dioxide incubator at 37 degrees Celsius for one to three weeks, allowing the cells to sit undisturbed for the first five days after the initial plating. After five days, replace the conditioned medium in each flask with 12 milliliters of fresh medium to achieve confluence. This must be done very carefully without touching the bottom of the flask where the cells attach.
Once the mixed glial cultures are completely confluence, remove the flask from the incubator. Cover the flask caps with param to prevent gas exchange with environmental air and place the flasks in a shaking incubator set to 100 RPS and 37 degrees Celsius for an hour after the hour has elapsed, use a 10 milliliter pipette to collect the media from the flasks without disrupting the astrocyte layer and dispense into 50 milliliter conical tubes. Add fresh media to the flasks and return to the incubator.
After centrifuging the tubes at 2, 500 RCF for five minutes of four degrees Celsius, aspirate the supinate and re suspend the cells in one milliliter of microglia plating medium. Then count the cells using a human cytometer and standard procedures. Once the number of cells has been determined, add an appropriate volume of microglial plating media to achieve a cell.
Concentration of two times 10 to the five cells per milliliter plate is appropriate for the experiment and return to the incubator. To allow the microglia to attach overnight. The cells have been immunostain with an antibody specific for IBA one, a microglial marker, which appears red.
There is minimal contamination of the culture with neuronal cells as demonstrated by this microscope image of immuno staining using an antibody for new NA neuronal marker, which appears red. The slide has been counters stained with DPI to stain all cell nuclei blue. This image shows immuno staining with GFAP and astrocyte marker.
The astrocytes appear green. Here we see an image of the microglial culture immuno stained with an antibody specific for CC one, an oligodendrocyte marker. The oligodendrocytes appear red.
This histogram shows the results of the quantification of each cell type. It can be seen that the plated microglial cultures are more than 90%pure. This fluorescence microscope image shows a microglial culture that has been immuno stain for IBA one.
The red areas are fluorescently labeled latex beads. The microglial culture shown here has been treated with one nanogram per milliliter lipo polysaccharide, and it can be seen that the microglial cells have phagocytosis the fluorescently labeled latex beads. Here, the results of the phagocytosis assay are displayed in a histogram.
Increased phagocytosis is seen after treatment with LPS. This figure shows that nitric oxide production also increases in microglial cultures after addition of LPS. Finally, both direct or transwell insert separated microglia neuron cultures are susceptible to increased cell death after incubation with LPS as measured by lactate dehydrogenase release Once mastered the first part of this technique, up until plating into T 75, flasks can be done in one and a half hours, and the complete procedure can be done within two weeks.
Another consideration during optimization of this protocol is to determine the duration of time. Mixed glial cultures should be maintained before isolating primary microglia for plating Following the establishment of high purity cultures. Using this procedure, microglia function can be assessed in vitro measurement of nitric oxide production, cytokine and chemokine release, as well as phasic activity can all be determined and measured using routine laboratory assays.
Thus, with the development of this procedure, researchers in the field of neuroscience have the capacity to explore microglia activity in a homogeneous cellular environment and investigate their roles under various neurological conditions. After watching this video, you should have a good understanding of how to prepare primary microglial cell cultures from neonatal rat brains. By first isolating the brain and removing the meninges, and then homogenizing the brain and plating into flasks, then the flasks are incubated for 10 to 14 days until an astrocyte monolayer has reached confluence.
The final step is shaking the flasks to release microglia that are growing on top of the astrocyte monolayer, resulting in a purified culture of microglia.
