Metal transport by Nramp1HA and Nramp2HA in CHO transfectants measured by quenching of calcein or Fura2 fluorescence. Metal transport was measured in independent transfected CHO cell lines stably expressing Nramp1HA (N1-94, N1-116, N1-123, N1-1816) or Nramp2HA (N2-310a) and in untransfected CHO controls (color coded and identified in the inset). For Fe²⁺ (A) and Co²⁺ (C) transport assays, cells were loaded with calcein-AM (0.25 μM), and the effect of extracellular Fe²⁺ (added as sodium ascorbate: Fe²⁺) or Co²⁺ on intracellular calcein fluorescence was continuously monitored at 37°C, pH 6.0, for 200 seconds (0.5-second intervals) using a Perkin-Elmer LS-50B fluorometer (excitation λ = 488 nm; emission λ = 517 nm; 5 μM bandpass slit width). For Mn²⁺ transport assays (B), cells were loaded with Fura2-AM (2 μM) and the effect of extracellular Mn²⁺ on intracellular Fura2 fluorescence quenching was monitored (excitation λ = 360 nm, emission λ = 510 nm, 7.5 μM bandpass slit width). Representative quenching curves are shown in panels A-C as relative fluorescence (Rel. Fluor.) normalized to the 70-second time point (immediately following addition of metal) for all groups. The average rate (with standard errors) of fluorophor quenching was calculated for Fe²⁺ (D), Mn²⁺ (E), and Co²⁺ (F) from the initial slope of 3-6 individual quenching curves. The mean quenching rates of all Nramp1/2HAtransfectants were statistically different (P < .005) than those of CHO controls with the following (still significant) exceptions: N1-116 (Fe²⁺; P = .010), N1-123 (Co²⁺; P = .05) as determined using the Student t test.