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Spectrofluorometric and FACS (Fluorescence Activated Cell Sorting) analyses were employed to determine 1) the maximal fluorescence excitation and emission peaks characteristic of BPD, benzoporphyrin derivative, 2) which structural analogue of BPD, BPD-monoacid ring A (BPD-MA), BPD-monoacid ring B (BPD-MB), BPD-diacid ring A (BPD-DA) or BPD-diacid ring B (BPD-DB) fluoresced to the greatest extent in the presence of leukemic cells and 3) to determine whether substantive differences existed in the uptake of BPD by human or murine leukemic versus normal human or murine mononuclear cells. Spectrofluorometric analysis revealed that the maximal fluorescence excitation peak of BPD (BPD-diacid ring A) was situated at 420 nm with a less prominent peak at 356 nm. Fluorescence emission scans, in which 420 nm was used as the excitation wavelength, revealed a single prominent fluorescence peak at 690 nm. FACS analysis revealed that negligible differences in fluorescence existed between leukemic cells incubated with BPD-MA, BPD-MB, BPD-DA, or BPD-DB upon excitation with visible light (488nm). However, subsequent to uv excitation cells incubated with BPD-MA fluoresced to the greatest extent followed by BPD-MB, BPD-DA, and BPD-DB respectively. Pronounced differences in red fluorescence were consistently observed between leukemic cells (HL60, K562, and L1210) and normal human or murine bone marrow cells incubated with BPD-MA. These observed differences in BPD-mediated fluorescence provide the rationale for sorting leukemic from normal cells via FACS and may constitute a novel method for extra-corporeal purging of remission marrow in autologous bone marrow transplantation.
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