2.8. Measurements of intracellular Ca2+

Intracellular calcium levels were monitored using the calcium-activated photoprotein, aequorin, as described previously [31]. In brief, HLMVECs were transduced with an adenovirus encoding aequorin at 30MOI, and 24 h later the aequorin was reconstituted by treating cells with 5 μM coelenterazine (Sigma, St. Louis, MO) for 30 min in serum-free and phenol free DMEM. The cells were then exposed to different concentrations of LLO, and luminescence was recorded using a PolarSTAR luminometer (BMG Labtech, Cary, NC).