Multi well plate

The murine macrophage cell line RAW264 (Riken, RCB0535) was used in the present study. Cells were cultured at 37°C under a humidified atmosphere of 5% CO₂–95% air in Eagle’s minimal essential medium (Nissui) containing kanamycin (60 μg/ml) and 10% (v/v) heat-inactivated fetal bovine serum (FBS, Biowest, Miami, FL). Cells were detached and seeded in each well of a multi-well plate (Becton, Dickinson and Company, Franklin Lakes, NJ) as described below.

The toxicity of the inhibitors on 1^st instar larvae was assessed using the protocol of Pridgeon et al. [22 (link)]. In brief, eggs were hatched in dH₂O under vacuum at room temperature for 2 h in a 250 ml beaker. The beaker was transferred to a rearing chamber (28°C) and 1^st instar larvae were collected 24 h later. For a given treatment, 5 larvae were transferred to the well of a 24 well Falcon MULTIWELL plate (Becton Dickinson Labware, Franklin Lakes, NJ) containing 995 μl of H₂O with a gap junction inhibitor and 5 μl of food solution. The food solution consisted of 0.013 g of ground Tetramin fish food (Tetra United Pet Group, Blacksburg, VA) suspended in 1 ml of dH₂O. Stock solutions of the inhibitors were dissolved in dH₂O and diluted within wells to achieve the desired concentrations (1 ml total volume per well). Plates were returned to rearing conditions and after 24 hours the larvae were assessed. The efficacy of a concentration was measured as the percentage of treated larvae in a well that were dead by 24 h. Larvae were considered dead if they did not move when disturbed with a 10 μl pipette tip.

Reagents were purchased from Sigma Chemical Co (St. Louis, MO, USA), tissue culture flasks, dishes, and multi-well plates were from Falcon Orange Scientific (Graignette Business Park, Belgium), culture media and supplements for both tissue and cell lines were from Gibco BRL (Carlsbad, CA, USA).