Isolation of microglia and border-associated macrophages

1. Mice were decapitated, brains were harvested and placed on ice-cold 2% FBS Iscove's Modified Dulbecco's Medium (IMDM).
2. Cut the large brain into small pieces. 
3. Then digest brain tissue in 5mL 2% FBS IMDM with 2U/mL DNase Ⅰ (Roche), 1 mg/mL Collagenase D (Roche) and 2U/mL Dispase Ⅱ at 270 rpm at 37 ℃ for 1 hour.
4. Then homogenize tissue with a syringe (19G needle) and the resulting homogenous cell suspensions were filtered through a 70 μm cell strainer.
5. Spin down at 350g for 5 minutes. Remove supernatant.
6. Cells were then resuspended in a 40% Percoll (GE Healthcare Life Sciences) and centrifuged at 700×g for 10 min (full brake).  (Or use 70% and 30% 2 layers Percoll, adjust the brake and collect the middle layer later instead)
7. Remove the supernatant, collect cell pellet (at the bottom), then resuspend in 0.89% NH₄CL lysis buffer for 5 min at room temperature (RT) to remove contaminating red blood cells (using 2 layer percoll can skip this step). 
8. Top up with IMEM medium, spin down at 350g for 5 min, the supernatant was discarded, and cell pellets were collected for following flow cytometry staining.
9. For microglia sorting, single cell suspension was pre-incubated with an-Fc receptor antibody for 20 min on ice. subsequently, cells were then stained with antibodies for 20 min on ice. After a washing step, cells were further stained with DAPI to exclude dead cells. Then the cells were ready for sorting.

To sort microglia, we use anti-CD45, CD11b, F4/80, P2RY12 to get CD45^intCD11b⁺F4/80⁺P2Ry12⁺ cell population.