2.2. Isolation and Culture of Primary Mouse Microglial Cells, Astrocytes, and Neurons

Primary cultures of mouse microglia were prepared from the whole brain of an embryonic day 17 (E17) mouse. Each embryo tail was used for genotype analyses to identify WT and STIM1^−/− microglia. Primary mixed cells that had been chemically and mechanically dissociated of the whole brain were seeded in DMEM/F12 (1 : 1) culture medium. Cells were cultured at 37°C in an incubator (Thermo Fisher Scientific, Marietta, OH, USA) humidified with 5% CO₂. Cell confluency was achieved after 3~4 weeks. After 4 weeks, the primary mixed cells of WT and STIM1^−/− were used a mild trypsinization and shaking method [19]. The upper cell layer of mixed cells was removed in one piece by treating with trypsin 0.25% (1x) solution (SH30042.01; Hyclone Laboratories, Thermo Scientific, Logan, UT, USA) diluted 1 : 4 in DMEM/F12 (1 : 1) culture medium (Dulbecco's modified Eagle medium, 11320-033; Gibco, Life Technologies™, Carlsbad, CA, USA) containing 10% fetal bovine serum (26140-079, Gibco, Life Technologies, Carlsbad, CA, USA) and 1% penicillin-streptomycin (15140-122; Gibco, Life Technologies, Carlsbad, CA, USA) for 20 min at 37°C. The trypsin diluted 1 : 4 in DMEM/F12 was suctioned and replaced with 12 mL of new culture medium. The remained pure microglial cells were attached to the bottom of the T75 flask and were isolated by shaking the T75 flasks of WT, STIM1^−/− at 120 rpm (SLOS-20; Seoulin Bioscience, Seoul, Korea) at 37°C for 1 h. 12 mL of each medium were collected from each flasks, placed a cell strainer with 40 μm pore size (352340; BD Falcon, San Jose, CA, USA), filtered into a fresh 15-mL conical tube, and centrifuged at 4,000 rpm at room temperature for 5 min. Glial cells were grown at a high density into 75 T flasks to extract pure microglia at 7 to 9 days in vitro. After isolate microglia, the remaining cells were maintained in astrocyte-conditioned medium and remaining microglia were depleted by adding 50 mM L-leucine-methyl ester (LME, Sigma Aldrich, St. Louis, MO, USA) for 4 hours. To culture the cortical neuron, the culture of cortical neuron was prepared from 1 day postnatal pups (C57BL/6 strain) as described previously [1] with some modifications. The cerebella were dissociated in Versene solution (1 : 5000) and plated at 0.5 × 10⁶ cells/cm² in 24-well plates coated with Laminin (10 μg/mL in serum-free DMEM). Cells were incubated in Dulbecco's modified Eagle's medium (DMEM) supplemented with heat-inactivated horse serum (5%), fetal calf serum (5%), 13 mM glucose, 0.5 mM HEPES buffer, 25 mM KCl, and 2 mM L -glutamine. Cells were maintained at in a humidified atmosphere of 37°C, 5% CO²/95% air. To inhibit growth of nonneuronal cells, 7.5 μM Ara-C (cytosine-D-arabinoside, Sigma Aldrich, St. Louis, MO, USA) was treated to the medium 48 h after plating. Cortical neuronal cells were used at 9 days after primary culture in experiment to ensure morphological and physiological maturity. Cells purities were checked with immunostaining of anti-GFAP antibody, anti-Iba1 antibody, and MAP antibody for glial cells, microglia, and neuron, respectively. The numbers of cells were counted with haemocytometer (Marienfeld, Lauda-Königshofen, Germany).