Charybdotoxin

Mouse EGF (Life Technologies) was diluted in fresh imaging buffer solution (3 mM Ca²⁺ in the extracellular medium, 3 mM Ca²⁺_o); 125 mM NaCl, 2.5 mM KCl, 1.1 mM NaH₂PO₄, 4 mM NaHCO₃, 2 mM MgCl₂, 3 mM CaCl_2, 10 mM glucose, 10 mM Hepes, 0.2% (W/V) BSA or in Ca²⁺ free buffer (nominally 0 mM Ca²⁺ in the extracellular medium, 0 mM Ca²⁺_o) solution; 125 mM NaCl, 2.5 mM KCl, 1.1 mM NaH₂PO₄, 4 mM NaHCO₃, 2 mM MgCl₂, 10 mM glucose, 10 mM Hepes, 0.2% (W/V) BSA, 1 mM EGTA (Sigma). EGF solutions of 40 pM and 4 nM (to be applied volume to volume to yield final concentrations of 20 pM and 2 nM, respectively) were kept on ice at all times and warmed up to 30°C just before stimulation to avoid degradation of EGF.
EGFR-specific neutralizing monoclonal M225 antibodies [12] (link) were chosen for their capacity to inhibit EGF binding, EGFR tyrosine kinase and proliferative activities and their use in cancer therapy (humanized version Cetuximab/C225). 40 µl of 100 µg/ml solutions of anti EGFR Ab-3 mouse M225 (Calbiochem, Merck Millipore) or IgG1 were added to 400 µl cell culture chamber 200 s after the start of time-lapse recording.
Charybdotoxin (Alomone labs), a blocker of Ca²⁺-activated K⁺ channels K_Ca1.1[13] (link), K_Ca3.1[14] (link) and voltage-dependent K_v1.3 channels [15] (link) was added to cells at a concentration of 100 nM, 20 min before starting the time-lapse recording.

1H‐[1,2,4]oxadiazolo‐[4,3‐a]quinoxalin‐1‐one (ODQ) was obtained from Enzo Life Sciences (Lausen, Switzerland) purchased through Eubio (Vienna, Austria). Apamin, charybdotoxin and iberiotoxin were purchased from Alomone Labs (Jerusalem, Israel). Collagenase (Worthington CLS 2) was from Worthington Biochemical Corporation (New York, USA). All other chemicals, including 5‐HMF, were of standard grade and purchased from Sigma‐Aldrich (Vienna, Austria).