2.11 Single-cell Ca2+ measurements
A549 cells were plated in 35 mm glass bottom dishes. After attachment, the cells were incubated with 2 µM Fura-2 AM (Beyotime, S1052) diluted in Ca2+ free HBSS for 30 min. The stained cells were sequentially treated with 800 µM PQ, 2 mM CaCl2, and lysine in a dose dependent manner, as indicated, for measurements of calcium fluxes. Fura-2 (340/380) filter set (pE-340fura, CoolLED, UK), a 20 × 0.3 N A. objective lens, and a Photometrics Iris9 camera was used to capture images at a frequency of 1 image pair every 2 s. Relative fluorescence ratio at wavelengths of 340 nM and 380 nM (F340/F380) was measured by Visview and utilized for the assessment of cytoplasmic calcium levels.