A. Culturing neurons
1. Dissect hippocampi from brains of E17-19 mice into cold Hanks. Remove all meninges.
2. Trypsinise (0.25% in Hanks). Stop with soybean trypsin inhibitor (1 mg/ml in Hanks).
3. Add small amount of complete medium (G3: Neurobasal + 2 ml B27 supplement + 25 uM Glu + 25 U Gln + 10 ug/ml gentamicin) and triturate using fire-polished pipette.
4. Plate onto pre-coated poly-L-lysine coverslips. Three-5 d later change the medium to G2 (G3 without Glu). Leave for 7 d, with a 50% change of medium every 3-4 d.
B. Culturing microglia
1. Dissect cortices from brains of E17-19 or P1 mice into cold Hanks. Remove all meninges.
2. Trypsinise. Stop with 1 ml/4 brains complete medium (DMEM + 10% FBS + 40 ug/ml gentamicin). Triturate with fire-polished pipette.
3. Plate onto pre-coated PLL flasks (I usually do 4 brains per T-75). Leave for 2 weeks, with a half change of medium every week.
4. Once the cell layers are stable and you start seeing rounded microglial cells on top of the layers (usually 10-14 d after the mixed cortical culture is plated), aspirate the medium off. You could also often harvest a lot of floating microglia at this time, so instead of asirating this off, collect and replate onto PLL-coated coverslips.
5. Add 12 mM lidocaine in Hanks (make a 60 mM stock and dilute 1:5) and let the flasks sit for about 5 min. Then gently shake the flask with a back-and-forth motion to help lift the microglia off. Constantly check under the microscope. Usually 3-5 min of shaking will be enough.
6. Collect the fluid and centrifuge to pellet the cells (very slowly!setting of 2 on clinical centrifuge, 10 min, cold).
7. Plate cells onto pre-coated PLL coverslips in complete medium. 15-30 min later, replace all of the medium. Culture for a few days until the cells look resting (long thin sticks).
8. Using sterile forceps, pick up the coverslips and place into inserts that are already in warm complete medium. I use the 6-well Nunc inserts (0.4 um pore).
9. Alternatively, you can just plate the microglia directly onto the inserts. The small pore size should be okay to exclude cell bodies.
10. Add LPS (and whatever other test substance) to the microglia, o/n. You might want to wash off the LPS before co-culture (remove 75% of CM and replace with fresh warmed medium).
C. Setting up the co-culture
1. Using sterile forceps, pick up the coverslips containing neurons and place into the bottom of the co-culture plate. Be sure to keep the cells covered with medium. Replace the insert with the microglia coverslip. Alternatively, you could change the medium in the microglial culture completely, and do a wash with fresh medium, before replacing the medium, to get rid of the LPS. Then add the neurons.
2. Incubate for 1 h. Then add Glu (I find that 25 uM is enough to begin killing neurons) to the bottom of the plate.
3. Three-five h later, collect conditioned medium, microglia (scrape off coverslip or insert membrane), and count neurons.
D. Microglial activation
1. Morphological changes: F4/80 or 5D4 antibody, isolectin B4 staining
2. NO release: measure nitrites formation by diaminonaphthalene
3. Nitrotyrosine formation on proteins
4. TNFa ELISA
E. Neuronal death
1. Live/Dead staining: propidium iodide/TOPRO-1 for membrane-impermeant, SYTO-13 for membrane-permeant (all cells)
2. LDH release