The effect of temperature on the recovery of fluorescence following addition of chelators in CALG-Fe-loaded endosomes of J774 cells. J774 cell endosomes were loaded under isotonic conditions with CALG-Fe (100 μM in DMEM for 20 minutes), then extensively washed with HBS medium containing 1% bovine serum albumin and perfused at either 37°C or 25°C with DMEM medium containing the indicated additives (100 μM chelators added as indicated) and probenecid (0.5 mM), which was supplemented in order to minimize leakage of the calcein. Images (epifluorescence) were taken at 1- to 2-minute intervals, and the average fluorescence intensity values of endosomes in 5 cells in a field were normalized to the corresponding one obtained at the onset of the treatment. (B) A fluorescent analog of DFO is taken up into endosomes of various cell lines. (i-ii) J774, (iii) H9C2, and (iv) HepG2 cells were incubated with 50 μM fluoresceinated DFO in growth medium for (i) 1 hour or (ii-iv) 12 hours, and after washing they were examined by LSM (fluorescein settings).