Flipr calcium 5

Neurons were incubated for 1h with the fluorescent calcium indicator FLIPR Calcium 5 (Molecular Devices R8186) diluted 3x more than providers recommendation in Hanks balanced salt solution (ThermoFisher Scientific, 14175) with 20 mM Hepes and 1.26 mM CaCl2. Image based measurements of cytosolic calcium kinetics were conducted at 37°C and with a 1 Hz sampling frequency (excitation and emission wavelengths of 480, and 540 nm, respectively) using the FDSS7000 system (Hamamatsu, Japan). The instrument readout is the total fluorescent intensity per well and data are reported as ratio to the first sampled data point.

Dimethyl sulfoxide (DMSO), N-formyl-Met-Leu-Phe (fMLF), phorbol 12-myristate 13-acetate (PMA), Trp-Lys-Tyr-Met-Val-Met (WKYMVM), Histopaque 1077, and viridiflorol were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Germacrone and β-phellandrene were purchased from TargetMol (Boston, MA, USA). Curzerene was purchased from ChemNorm (Wuhan, China), and spathulenol was purchased from ChemFaces (Wuhan, China). n-Hexane was purchased from Merck (Darmstadt, Germany). Fluo-4AM was purchased from Invitrogen (Carlsbad, CA, USA), and FLIPR Calcium 5 was from Molecular Devices (Sunnyvale, CA, USA). Roswell Park Memorial Institute (RPMI) 1640 medium and Dulbecco’s Modified Eagle’s Medium (DMEM):F12 medium were purchased from HyClone Laboratories (Logan, UT, USA). Fetal calf serum and fetal bovine serum (FBS) were purchased from ATCC (Manassas, VA, USA). Hanks’ balanced salt solution was purchased from Life Technologies (Grand Island, NY, USA). HBSS without Ca²⁺ and Mg²⁺ is designated as HBSS^–; HBSS containing 1.3 mM CaCl₂ and 1.0 mM MgSO₄ is designated as HBSS⁺.