Isolation and Culture of Microglia from Rat Brains using Serum-Free Media

Take a perfused rat brain. Mince it into small fragments.

Homogenize the tissue in a buffer to release cells, including microglia.

Once the undigested tissue settles, collect the supernatant containing cells and add a myelin separation buffer.

Mix the contents and centrifuge to separate cells from myelin and debris. Remove the myelin-debris layer and supernatant.

Resuspend cells in buffer and pipette to separate clumps.

Pass the suspension through a strainer to remove remaining cell aggregates.

Transfer the filtered cells to a culture plate coated with microglia-specific monoclonal antibodies. Incubate to facilitate microglia adhesion.

Wash with buffer to remove non-adherent cells. Add trypsin and incubate to loosen microglia.

Remove trypsin.

Add serum-free media. Incubate on ice to weaken cell-substrate interactions.

Pipette and collect the suspension in a tube. Centrifuge and remove the supernatant.

Resuspend microglia in serum-free media and transfer onto a collagen-coated multi-well plate. Incubate to facilitate microglia adhesion.

Add media with growth factors and lipids. Incubate for microglia maturation.

After the brains have been collected in cold DPBS, chop each brain into one-cubed-millimeter chunks in a Petri dish on ice with a cold scalpel blade, and transfer to an ice-cold Dounce homogenizer with 5 to 7 milliliters of ice-cold douncing buffer. Next, use a loose-fitting Dounce homogenizer to dissociate the tissue with 10 to 20 gentle and incomplete strokes. Take care not to directly crush the tissue at the bottom of the homogenizer. Instead, impel the tissue through the space between the sides of the piston and the homogenizer.

It is important to slowly homogenize the tissue with multiple rounds of douncing to preserve microglia viability.

Afterward, carefully remove the piston to prevent introducing air bubbles. Allow poorly-dissociated tissue chunks to settle to the bottom of the homogenizer, and transfer the supernatant to a new, chilled 50-milliliter conical tube. Subsequently, replace the removed volume with fresh douncing buffer, and repeat the dissociation procedure for a total of three to four rounds, or until all tissue has been dissociated.

In this procedure, add ice-cold douncing buffer to the 50-milliliter conical tube containing the cell suspension, and adjust the total volume to 33.5 milliliters. Next, add 10 milliliters of MSB to the cell suspension, and mix thoroughly by inverting the tube several times. This will result in a 23% final concentration of MSB in a volume of 43.5 milliliters.

After that, centrifuge the cells for 15 minutes at 500 g at 4 degrees Celsius with slow braking. Then, remove the top layer of myelin, and debris, and the supernatant with a pipette. Take care when removing the top layer to ensure as much of it is removed as possible.

Following that, resuspend the cell pellet in 12 milliliters of panning buffer. Gently triturate the cell suspension, to break up any remaining clumps of cells. In this step, pass the cell suspension through a 70-micrometer cell strainer to remove large debris or cell clumps. Rinse the OX42-coated panning dish three times with DPBS. Don't allow the plate to completely dry between washes.

Pour off the last DPBS wash and apply the filtered cell suspension to the panning dish. Gently swirl the plate to distribute the cells. Then, incubate the plate on a flat surface at room temperature for 20 minutes to allow cells to adhere. Do not incubate longer than 20 minutes, or cells will become very difficult to recover from the dish. Afterward, rinse the panning dish with DPBS 10 times to remove non-adherent cells.

Microglia will be firmly attached to the plate, so swirl the plate with each rinse to ensure the removal of other non-adherent cells. Pour off the last DPBS wash, and replace with 15 milliliters of DPBS and 200 microliters of trypsin.

To trypsinize the cells, incubate the dish for less than or equal to 10 minutes at 37 degrees Celsius. After 10 minutes of trypsinization, microglia will still be stuck to the plate. Pour off the mixture and gently wash the plate two times with DPBS to remove the trypsin. Then, replace with 12 milliliters of ice-cold MGM.

Subsequently, place the panning dish on ice for two minutes to help weaken cell and substrate interaction, and make sure that the dish is flat to prevent areas of the panning dish from drying out. After that, pipette vigorously with a 10-milliliter pipette and pipette controller on high speed to recover cells from the panning dish. Draw a 16-by-16 grid with the stream of liquid from the pipette to try and remove all of the cells.

It is important to find a balance between overall cell recovery and the health of your culture. Repeated pipetting of a cell supernatant against the panning dish will result in reduced viability of your culture.

Check the cells under a microscope at 20x magnification to make sure that they have detached from the plate. Mark the spots on top of the dish where cells are stuck and repeat pipetting in those areas. Collect the cell suspension and aliquot 3 to 4 milliliters of supernatant in each 15-milliliter conical tube. Spin it for 15 minutes at 500 g at 4 degrees Celsius with slow braking.

After 15 minutes, aspirate the supernatant, leaving 0.5 milliliters of MGM with the cell pellet. Then, resuspend each pellet in the remaining MGM and pool the cells from all the tubes. Following that, count the cells with the hemocytometer.

Now, plate 15 microliters of collagen IV coating directly in the center of a 24-well anionic, cationic-coated tissue culture plate, and incubate for 15 minutes at 37 degrees Celsius. After counting the cells with a hemocytometer, dilute them to 2.3 x 10^-5 per milliliter in MGM. Aspirate collagen IV spot, and immediately plate 15 microliters of cell suspension to this spot to yield 3.5 x 10³ cells per spot.

Next, incubate for five to ten minutes at 37 degrees Celsius to allow the cells to adhere. Then, gently add 500 microliters of CO₂-equilibrated TIC to the well.