Short-term recovery of LIP levels in iron-depleted K562 cells. K562 cells were incubated in HBS (2 × 10⁶/mL) alone or in the presence of 100 μmol/L SIH for 10 minutes at 37°C to examine acute iron depletion. Cells were washed free of chelator and incubated for an additional recovery period either in Dulbecco’s modified Eagle’s medium (DMEM) alone or DMEM supplemented with 20 μg/mL leupeptin, 50 μg/mL transferrin, or 10% FCS. At the end of the incubation period, the cells were washed with HBS and processed for LIP measurements by loading with calcein-AM (CA), washed, and resuspended in HBS-medium. Fluorescence (left panels) was continuously monitored as described in Materials and Methods. When fluorescence readings were steady, anti-CA antibodies (ab) were added (to quench extracellular fluorescence), followed by 100 μmol/L SIH, to attain maximum recoverable fluorescence. Shown are tracings obtained after recovery periods of 0 to 6 hours (ISAH-0-6 h). The maximum value, which was equivalent to the concentration of CA-bound iron [CA-Fe], was used for calculating LIP values (in micromoles per liter). LIP values are shown in the right panels for the different treatments: (A) recovery in DMEM alone (recov), (B) recovery in DMEM supplemented with leupeptin (recov.+leup), or (C) recovery in DMEM supplemented with transferrin (Recov. + Tf) or FCS (Recov.+ FCS).