Calcein Loading of Cells and Iron Influx Assay

Calcein-AM is a membrane-permeable, non-fluorescent molecule that becomes fluorescent upon intracellular cleavage by cytoplasmic esterases to calcein (which is membrane impermeable). It is pH-independent and is rapidly quenched by divalent metals and reversed easily by their chelators. Ferrous iron influx into VM neurons was determined by the quenching of calcein fluorescence as described previously [31]. The VM neurons were divided into 4 groups. Control: VM neurons were cultured in serum-free DMEM/F12 supplemented with 2% B27 for 24 h; MPP⁺: VM neurons were cultured in serum-free DMEM/F12 supplemented with 2% B27 with 5 μmol/L MPP⁺ for 24 h; MPP⁺ with isradipine or Bayk8644: MPP⁺-treated cells underwent the same procedures except that isradipine or 10 µmol/L Bayk8644 was included in the perfusion fluid. The cells were incubated with calcein-AM at a final concentration of 1 µmol/L in HBS for 30 min at 37 °C. Excess calcein on the cell surface was washed 3 times with HBS. Calcein fluorescence was recorded at 488 nm excitation and 525 nm emission. Fluorescence intensity was measured every 3 min for the next 30 min during perfusion with 100 µmol/L ferrous iron (FeSO₄ in ascorbic acid solution, 1:44 molar ratio, pH 6.0). The mean fluorescence intensity of 30–35 cells in 4 separate fields was monitored at 200× magnification and processed with Leica Application Suite X (Leica, Mannheim, Germany).