Poly d lysine

Stable inducible clones of US28 and CX₃CR1 cells were seeded at 10,000 cells/well in poly-D-lysine (Invitrogen) coated 96-well tissue culture plates (Nunc). One day after seeding US28 and CX₃CR1 receptor expression was induced by tetracycline (Invitrogen; 3,6 ng/mL and 5 ng/mL, resp.) aimed at obtaining 5–10% specific binding of the added radioactive ligand. One day after induction, cells were assayed by competition binding for 3 h at 4°C using 20–70 pM ¹²⁵I-CX₃CL1 as well as unlabeled ligand 10 pM to 100 nM in 50 mM Hepes buffer pH 7.4, supplemented with 1 mM CaCL₂, 5 mM MgCL₂, and 0,5% (w/v) bovine serum albumin (BSA) (binding buffer). After incubation, cells were washed twice in ice-cold binding buffer and supplemented with 0,5 M NaCl. Determinations were made in quadruplicate.

All mice were handled in accordance with the guideline for animal care and use of the Yonsei University. All experimental procedures were approved by the Institutional Animal Care and Use Committee of the Yonsei University (permissions IACUC (2017-10-647-01 and 2018-01-689-01)). Cerebral cortices were removed from gestational day 14.5 mouse embryos (Orient, Gyeong-gi, Republic of Korea) and mechanically dissociated as previously described^{79 (link)}. Briefly, dissociated cortical cells were plated at a density of 5×10⁶ cells per well of six-well plates or at 1×10⁶ cells per well of 24-well plates that were precoated with 100 μg/mL poly-d-lysine and 1 μg/mL laminin (Invitrogen, Carlsbad, CA, USA). Cultures were incubated at 37 °C in Minimum Essential Medium (MEM, Gibco) supplemented with 0.6% glucose (Sigma-Aldrich), 1 mM sodium pyruvate (Sigma-Aldrich), 2 mM l-glutamine (Sigma-Aldrich), penicillin-streptomycin (100 U/mL), and 10% FBS (Gibco) in the atmosphere of 95% air and 5% CO_2. After 24 h, culture medium was changed to Neurobasal medium (Invitrogen) supplemented with 2% B-27 (Gibco), 0.5 mM l-glutamine and 10 μM cytosine β-d-arabinofuranoside (Ara-C, Sigma-Aldrich). At 4 DIV, cultures were treated with the indicated drugs that were dissolved in the same medium.

Stable inducible clones of the US28-cells were seeded in poly-D-lysine (Invitrogen) coated 96-well tissue culture plates (clear plates, Costar). The number of cells seeded was determined by the apparent expression of receptors and was aimed at obtaining 5–10% binding of the added radioactive ligand. US28 receptor expression was induced by tetracycline one day after seeding the cells (0.25 µg mL⁻¹; Invitrogen). 48 hr post seeding, cells were washed twice in ice-cold binding buffer (50 mM HEPES pH 7.4, supplemented with 1 mM CaCl₂, 5 mM MgCl₂ and 0.5% (w/v) bovine serum albumin) and assayed by competition binding for 3 hr at 4˚C using 10–15 pM ¹²⁵I-CX3CL1, ¹²⁵I-CCL5, or ¹²⁵I-CCL3 as well as unlabeled ligand (10 pM to 100 nM in binding buffer). After incubation, cells were washed twice in ice-cold binding buffer, supplemented with 0.5 M NaCl.

Primary hippocampal neurons were cultured from postnatal day 0 (P0) C57BL/6 mice. Bilateral hippocampi were dissected, and the hippocampal tissue was washed twice with PBS and digested with 0.25% trypsin (Gibco, CA, USA) for 10 minutes. An equal volume of DMEM-F12 medium (Gibco, CA, USA) with 10% fetal bovine serum (FBS) (Gibco, CA, USA) was added to stop digestion. The supernatant was collected and centrifuged at 1000 rpm for 5 minutes, the resulting supernatant was discarded, and implant solution (DMEM-F12 medium with 10% FBS) was added. The cell suspension was dripped onto a cover slip coated with poly-D-lysine (Invitrogen, CA, USA) and incubated at 37° C. After half of an hour, neurobasal medium (Gibco, CA, USA) with Pen-Strep (Invitrogen, CA, USA), B27 (Gibco, CA, USA) and GlutaMAX (Thermo Fisher, MA, USA) was added. Si-055714, si-NC, miR-7684-5p inhibitor and miR-NC were obtained from Generalbiol (Chuzhou, China). Neurons were transfected with Lipofectamine™ 2000 (Invitrogen, CA, USA) on DIV4.