This protocol allow us to culture microglia in conditions that closely mimics the in vivo characteristics. Using magnetic cell sorting technology, our protocol allow us to stimulate microglia only two day in vitro, without using any serum in the culture medium. To begin, use small scissors to cut the skin from the neck to the nose, following the sagittal suture of 15 to 20 millimeters.
Insert the tips with the foramen magnum parallel to the skull. Cut from each side to the eyes. Cut between the eyes with small scissors to detach the skull and brain from the head.
With two forceps, grab the skull close to the olfactory bulbs, and carefully tear the skull. With a razor blade, remove the cerebellum and olfactive bulb, and cut the brain into two pieces. Place the brain pieces in a Petri dish containing 40 milliliters of HBSS without calcium and magnesium.
Prepare dissociation mixture according to this table. Transfer 12 brain pieces into a C tube for a total weight of 1.2 grams per dissociation tube. Then place C tubes on the dissociater with heating.
Start the optimized NTDK program in the dissociater. Centrifuge for 20 seconds. Complete the mechanical dissociation by pipetting three times.
Transfer the cells to four 15-milliliter tubes with attached strainers. Rinse the strainers with 10 milliliters of HBSS with calcium and magnesium. Centrifuge for 10 minutes, and remove the supernatant with a 10-milliliter pipette.
Carefully add 10 milliliters of HBSS with calcium and magnesium, and re-suspend the pellet. Again, centrifuge and remove the supernatant. Re-suspend the pellet with six milliliters of sorting buffer.
Repeat the centrifugation and discard the supernatant. Then add 200 microliters of CD11b microbead solution and incubate the tubes for 15 to 20 minutes at four degrees Celsius. After incubation, re-suspend the pellet with six milliliters of sorting buffer.
Repeat the centrifugation and re-suspend the pellet with eight milliliters of sorting buffer. Next, follow the Possel program on the separator to prepare eight columns. Pass the cells through the column by adding one milliliter of cell suspension at a time.
With one milliliter of sorting buffer, elute CD11b-positive cells on a sterile elution plate. Pool the cells in a 50-milliliter tube. Centrifuge and re-suspend the pellet with 10 milliliters of cold microglia medium.
Count the CD11b-positive cells. Re-suspend the cells in cold microglia medium to get a final concentration of 650, 000 to 700, 000 cells per milliliter. Dispense the suspension in cell culture plates.
Incubate the plates overnight at 37 degrees Celsius with 5%carbon dioxide. On the next day, replace the medium with preheated microglia medium, and repeat the overnight incubation. Stimulate the cells before this step, and perform phagocytic assay during the last three hours of the stimulation.
Calculate the number of beads according to this table at a ratio of 50 beads per cell. Prepare the beads mixture. Incubate the tube for one hour in a water bath at 37 degrees Celsius.
Vortex every 10 minutes. To each well, add the calculated volume of the bead solution and incubate for three hours. Using this approach, the phagocytic activity of microglia was evaluated after stimulation by pro-inflammatory factors, such as interleukin-1 beta, plus interferon gamma, or lipopolysaccharides.
After stimulation for six to 24 hours, fluorescent Cy3 microglia beads were analyzed by confocal microscope. After six hours, microglia start to phagocyte Cy3 beads only under interleukin-1 beta plus interferon gamma condition. After 24 hours, there was an increase of Cy3 fluorescence for both kinds of stimulation, highlighting increased phagocytic activity.
The purity of microglial culture was elevated by flow cytometry, which showed an increase in cell viability after sorting. Using flow cytometry and RT-qPCR cell population markers were analyzed before and after sorting CD11b-positive cells from mice brains at postnatal day eight. These analyses distinguished various brain cell populations, such as CX3CR135 for microglia, O4, or OLIG2 for oligodendrocytes, NeuN, or synaptophysin, for neurons, and ACSA-2, or GFAP for astrocytes.
After cell sorting using CD11b antibody, only microglia were obtained. It is important to pipette very gently while adding the suspension to the column to prevent clogging and avoid mechanical cell death. After this method, transcriptomic proteomics, such as western blood, or Luminex, and even immunochemistry can be performed in order to see the effect of microglial stimulation.
