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

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

1. Prepare a Petri Dish for CD11b Immunopanning (Day 0)

NOTE: Prepare 1 immunopanning dish for every 1 – 2 juvenile rat brains.

1. Add 25 mL of 50 mM Tris pH 9.5 solution to a 15-cm Petri dish.
2. Add goat anti-mouse IgG (H+L chains) to the dish for a final concentration of 6 µg/mL. Swirl plate to evenly distribute.
3. Incubate the dish for 1 – 3 h at 37 °C.
4. Rinse dishes three times with DPBS++ (phosphate-buffered saline (PBS) with Ca²⁺ and Mg²⁺), then replace with a solution of panning buffer containing 1 µg/mL OX42 antibody. Leave the dishes overnight at room temperature on a flat surface.

2. Tissue Collection (Day 1)

NOTE: This protocol should take ~3 – 4 h.

1. Before beginning, ensure all solutions are sterile and chilled on ice. Chill all instruments on ice and sterilize with ethanol prior to use.
2. Following appropriate regulatory procedures, sacrifice a juvenile laboratory rat by carbon dioxide asphyxiation.
   NOTE: Alternatively, younger animals may be sacrificed with a lethal dose of ketamine/xylazine (100 – 200 µL of 24 mg/mL ketamine, 2.4 mg/mL xylazine). Ketamine/xylazine must be used if animals are less than 14 days of age. If using multiple animals, extract the tissue from one animal and place it into pre-chilled DPBS⁺⁺ on ice before proceeding to subsequent animals.
3. Pinch a hindpaw and ensure complete unresponsiveness from the animal before proceeding.
4. Transcardially perfuse the animal with 10 – 30 mL of ice-cold perfusion buffer using a 27½-G needle until buffer runs clear.
   NOTE: Volume of Perfusion buffer will vary depending on the size/age of the animal.
5. Immediately after perfusion remove the head of the animal. Coming in from the spinal cord cut the occipital condyle on each side with dissection scissors, be careful not to damage the brain. After cutting each side carefully, cut up one side along the parietal and frontal bone towards the nasal bone. With forceps carefully pull back the top of the skull, quickly remove the brain (or CNS structures of interest), and place into pre-chilled DPBS⁺⁺ on ice.
6. Repeat for all remaining animals.
   NOTE: All proceeding steps should be performed in a laminar flow hood under proper sterile conditions.

3. Mechanical Dissociation (Day 1)

1. After all brains have been collected, chop one brain into 1 mm³ chunks in a Petri dish on ice with a cold scalpel blade, and transfer to an ice-cold dounce homogenizer with 5 – 7 mL ice-cold douncing buffer. Dissociate one brain at a time.
2. Dissociate the tissue using 10 – 20 gentle and incomplete strokes with a loose-fitting dounce homogenizer. Take care not to directly crush the tissue at the bottom of the homogenizer, but instead impel the tissue through the space between the sides of the piston and the homogenizer.
3. Carefully remove the piston to prevent introduction of air bubbles. Allow poorly dissociated tissue chunks to settle to the bottom of the homogenizer, and transfer supernatant to a new chilled 50-mL conical tube.
4. Replace the removed volume with fresh douncing buffer, and repeat steps 3.2 and 3.3 for a total of 3 – 4 rounds, or until all tissue has been dissociated. Repeat the procedure for each brain.

4. Myelin Removal (Day 1)

NOTE: Myelin removal is used for isolation of microglia from animals older than P12.

1. Measure the volume of the cell suspension in the 50-mL conical tube using a 25 mL pipette, then add ice-cold douncing buffer to adjust the total volume to 33.5 mL.
2. Add 10 mL of myelin separation buffer (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 mL.
3. Centrifuge cells for 15 min at 500 x g at 4 °C with slow braking.
   NOTE: The centrifuge should take approximately 1.5 – 2 min to decelerate. This will generate an upper layer of myelin and dead cell debris, a somewhat murky supernatant, and a smaller pellet that is enriched for live cells.
4. Remove the top layer of myelin/debris and the supernatant with a pipette. Take care when removing the top layer to ensure as much of it is removed as possible.
5. Resuspend the cell pellet in 12 mL of panning buffer. Gently triturate the cell suspension to break up any clumps of cells that might remain.

5. Immunopanning (Day 1)

1. Pass the cell suspension through a 70-µm cell strainer to remove large debris or cell clumps.
2. Rinse the OX42-coated panning dish three times with DPBS++. Don't allow the plate to completely dry between washes.
3. 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 min to allow cells to adhere. Do not incubate longer than 20 min or cells will become very difficult to recover from the dish.
4. 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 removal of other non-adherent cells.
5. Pour off the last DPBS⁺⁺ wash and replace with 15 mL DPBS++ and 200 μL trypsin (1.25% stock solution).
6. Incubate the dish for ≤10 min at 37 °C with 10% CO₂ to trypsinize.
   NOTE: Do not continue longer than 10 min or microglia will become difficult to remove.
7. After 10 min of trypsinization, microglia will still be stuck to plate. Pour off trypsin/DPBS⁺⁺ and gently wash 2x with DPBS⁺⁺ to remove trypsin, replace with 12 mL of ice-cold microglia growth medium (MGM).
8. Place panning dish on ice for 2 min to help weaken cell/substrate interaction, and make sure the dish is flat to prevent areas of the panning dish from drying out.
9. Pipette vigorously with a 10-mL pipette and pipet controller on high speed to recover cells from the panning dish. Draw a 16 x 16 grid with the stream of liquid from the pipette to try and remove all the cells.
10. Check cells under a microscope at 20X magnification to make sure cells have detached from plate. Mark spots on top of the dish where cells are still stuck, and repeat pipetting in those areas.
11. Collect cell suspension and aliquot 3 – 4 mL of supernatant per 15-mL conical tube. Spin for 15 min at 500 x g at 4 °C with slow braking.
    NOTE: Spinning microglia through a small volume allows for the maximum recovery of cells.
12. Aspirate the supernatant, leaving 0.5 mL of MGM with the cell pellet.
13. Resuspend each pellet in remaining MGM, and pool the cells from all the tubes.
14. Count cells with hemocytometer.
15. Plate cells as described in Steps 6 – 7 depending on the application.
16. Culture cells at 37 °C with 10% CO₂.
    NOTE: Cells can be culture for up to 3 – 4 weeks with regular media changes or one week with no media changes.

6. Spot Coating Tissue Culture Plates/Coverslips (Day 1)

1. Plate 15 µL of collagen IV coating directly in the center of a 24-well anionic/cationic coated tissue culture plate (see Table of Materials for details) and incubate for 15 min at 37 °C with 10% CO₂.
2. After counting cells with hemocytometer dilute cells to 2.3 x 10⁵/mL in MGM. Aspirate collagen IV spot and immediately plate 15 µL of cell suspension to this spot; this will give 3.5 x 10³ cells/spot. Incubate for 5 – 10 min at 37 °C with 10% CO₂ to allow cells to adhere, add 500 µL of CO₂-equilibrated TGF-β2/IL-34/cholesterol containing growth medium (TIC) gently to the well.