HbF in normal erythroid cultures: HPLC and fluorescence-activated cell sorter (FACS) analyses. (A) HPLC analysis. Cultures of cells derived from healthy individuals were either untreated (□) or treated with 150 μMHU(▦)or20nM MTH (▪). The drugs were added to the cultures on day 4 of phase 2 and the cells harvested on day 12. The cells were washed, lysed, and the hemolysate analyzed for hemoglobins by HPLC. The results present the %HbF (mean values ± SD of independent induction experiments performed on precursors from 5 healthy individuals; P < .001 when data from MTH- or HU-treated cell samples are compared with control; P < .005 when MTH-treated and HU-treated samples are compared). (B) Effects of timing of addition of MTH on cell growth and HbF production. MTH (at 0, 10, and 15 nM) was added to erythroid cultures on either the first (▪) or the fourth (▦) day of phase 2. □ indicate control untreated cells. On day 13, cells were harvested, an aliquot was counted by benzidine staining, and the rest of the cells were washed, lysed, and analyzed for hemoglobins by HPLC. Left panel: %HbF out of the total Hb produced. Right panel: number of erythroid cells per milliliter (× 10^–6). The results represent mean values ± SD of 3 independent experiments. (C) FACS analysis. A culture aliquots were permeabilized, stained with antihuman HbF antibodies, and analyzed by flow cytometry. Dot plots of forward light scatter (FSC) and phycoerythrin (PE) fluorescence of 10 000 cells are shown. The horizontal lines denote the level of fluorescence of cells stained with an isotype control antibody.