Oxotremorine m

Ca²⁺ measurements were performed essentially as described previously [20 (link), 78 (link), 79 (link)]. SH-SY5Y cells were loaded with 2 μM Fluo4-AM and/or Rhod2-AM dye (Invitrogen) in external solution (145 mM NaCl, 2 mM KCl, 5 mM NaHCO₃, 1 mM MgCl₂, 2.5 mM CaCl₂, 10 mM glucose, 10 mM Na-HEPES, pH 7.25) containing 0.02% Pluronic-F27 (Invitrogen) for 15 min at 37 °C, followed by washing in external solution for 15 min at 37 °C. Fluo4 and Rhod2 fluorescence were timelapse recorded (1 s intervals) at 37 °C using either an Axiovert S100 microscope (Zeiss) driven by MetaMorph (Molecular Dynamics) and equipped with GFP (Fluo4) and DsRed (Rhod2) filtersets (Chroma Technology), a 40× Plan-Neofluar 1.3NA objective (Zeiss), and a Photometrics Cascade-II 512B36 EMCCD camera or a Nikon Ti-E microscope using a CFI Plan Apo VC 20× objective and Nikon Andor Neo sCMOD high-resolution camera and appropriate filter sets. The cells were kept under constant perfusion with external solution (0.5 ml/min). Inositol 1,4,5-trisphosphate (IP3) receptor-mediated Ca²⁺ release from ER stores was triggered by application of 100 μM Oxotremorine-M for 2 min. Ca²⁺ levels were calculated as relative Rhod2 or Fluo4 fluorescence compared to baseline fluorescence (F/F₀) at the start of the measurement. Oxotremorine-M was from Santa Cruz Biotechnology and was dissolved in water.

B. malayi and Brugia pahangi adults extracted from the Meriones unguiculatus infection system (NIH/NIAID Filariasis Research Reagent Resource Center) (105 (link)) were maintained in daily changes of RPMI 1640 medium with l-glutamine (Sigma-Aldrich) supplemented with fetal bovine serum (FBS) (10%, vol/vol; Fisher Scientific) and penicillin-streptomycin (0.1 mg/mL; Gibco) at 37°C with 5% CO₂ unless otherwise specified. Brugia microfilariae isolated from the same system were maintained in RPMI 1640 medium supplemented with l-glutamine (Sigma-Aldrich) and penicillin-streptomycin (0.1 mg/mL; Gibco) at 37°C with 5% CO₂ unless otherwise specified.
The chemicals used in the assays included serotonin (catalog number AAB2126306; Fisher Scientific), arecoline (catalog number AC250130050; Fisher Scientific), atropine (catalog number sc-252392; Santa Cruz Biotechnology), nicotine (catalog number sc-482740; Santa Cruz Biotechnology), levamisole (catalog number TCL0231-1G; VWR), carbachol (catalog number sc-202092; Santa Cruz Biotechnology), acetylcholine (catalog number AC159170050; Fisher Scientific), oxotremorine M (catalog number sc-203656; Santa Cruz Biotechnology), and aldicarb (catalog number sc-254939; Santa Cruz Biotechnology).