Clodronate

2-Arachidonylglycerol (2-AG, 10 nM, stock solved in DMSO; Tocris, Minneapolis, MN, United States, cat No. 1298), Clodronate (100 μg/ml, stock solved in Aqua; Bayer Vital GmbH GB; PZN: 04299668), Palmitoylethanolamide (PEA, 10 nM, stock solved in DMSO, Tocris, cat No. 0879), LPS (10 ng/ml, stock solved in Aqua; Sigma-Aldrich, cat No. L8274) and NMDA (50 μM, stock solved in Aqua bidest., Sigma-Aldrich, cat No. M3262) were used and applied to the culture medium according to treatment protocol.

Primary microglia were detached from microglia–astrocyte co-cultures prepared from cerebral cortices of neonatal wild-type mice as described before (Grabiec et al., 2019). Brains were removed, and cells were dissociated after treatment with 4mg/mL trypsin (Merck Millipore, Burlington, MA, USA) and 0.5 mg/mL DNAse (Worthington, Bedford, MA, USA) in Hank’s balanced salts solution (Invitrogen, Carlsbad, CA, USA). This procedure resulted in the growth of a confluent astrocyte monolayer with attached microglia cells on top. Microglia cells were isolated from the monolayer by gentle shaking, and a purity of approximately 99% was reached.
Murine primary cells were cultured in medium consisting of DMEM (Invitrogen) with 10% FBS (Invitrogen) and 1 mL streptomycin/penicillin.
The microglial cells (5.000) were seeded into 24-well plates and treated with nimodipine (Bayer, Leverkusen, Germany, 0.1 µM, solved in ethanol), nifedipine (Bayer, 0.1 µM, solved in ethanol) or clodronate (10 µg/mL solved in water, Bayer for 24 h). For cell death analyses, propidium iodide (PI, 5 µg/mL, Sigma Aldrich, St. Louis, MO USA) was added 2 h before the fixation with 4% paraformaldehyde (PFA).
Cells were incubated with nucleic acid stain Sytox Green (Invitrogen, 1:10.000) and covered with DAKO fluorescent mounting medium (DAKO Diagnostika GmbH, Hamburg, Germany).