Polycarbonate filter

Monoclonal IgE (clone SPE7), RPMI 1640, 2-mercaptoethanol (2-ME), Laemmli buffer, L-α-lysophosphatidylinositol (LPI), and sphingosine-1-phosphate (S1P) were purchased from Sigma-Aldrich (St. Louis, MO, USA). HEPES, non-essential amino acids (NEAA), penicillin, streptomycin, and fetal bovine serum (FBS) were obtained from Gibco-BRL-Life Technologies (Gaithersburg, MD, USA). Interleukin (IL)-3 was purchased from PeproTech (Rocky Hill, NJ, USA). The GPR55 synthetic agonist, O-1602, was purchased from Cayman Chemical (Ann Arbor, MI, USA), while the GPR55 antagonist, ML-193, was purchased from TOCRIS (Bristol, UK). The CB2 antagonist, AM630, was from Sigma-Aldrich (St. Louis, MO, USA). Anti-p-cofilin (pSer 3) antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-p-LIMK (pThr 508/505) and anti-β-actin (C-terminal) antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The secondary antibodies, HRP-coupled anti-mouse and anti-rabbit, were obtained from Jackson ImmunoResearch (West Grove, PA, USA). Rhodamine-labeled phalloidin and calcein-AM were obtained from Life Technologies (Carlsbad, CA, USA). DAPI was purchased from Invitrogen (Carlsbad, CA, USA). The polycarbonate filters were from Neuro Probe, and bovine skin gelatin from Sigma-Aldrich (St. Louis, MO, USA)

The effect of the compound on directed migration and general motility was tested using a 48-well micro chemotaxis Boyden chamber (Neuroprobe, Gaithersburg, USA). Upper and lower wells were separated by polycarbonate filters (8 μm pore size; Neuroprobe, Gaithersburg, USA). Microglial cells (2–4 × 10⁴ cells) and 50-μL plain medium were added to the upper compartment. In addition, 2.5 μM of compound in DMSO, 125x10^-5 v/v DMSO respectively, and/or 100 μM ATP were added to the upper and/or lower chamber, as shown in Fig 3A and 3B. Plain medium was used as a control. The chamber was incubated at 37°C and 5% CO₂ for 6 h. Cells remaining on the upper surface of the membrane were removed by wiping, and cells in the lower compartment were fixed in methanol for 10 min and subjected to Diff-Quik stain (Medion Grifols Diagnostics AG, Düdingen, Switzerland). The rate of microglial migration was calculated by counting cells in four random fields of each well using a 20× bright-field objective. All data were normalized to the ATP-induced chemotaxis.