Novel multiparameter flow cytometry assay using Syto16 for the simultaneous detection of early apoptosis and apoptosis‐corrected P‐glycoprotein function in clinical samples

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Abstract
Background The fluorescent probe Syto16 has been used successfully to measure P‐glycoprotein (Pgp) function and, separately, early apoptosis and cell death. The present study was designed to evaluate whether the combined use of Syto16, the Pgp blocker PSC833, and 7‐AAD allows simultaneous detection of all parameters, with emphasis on applications in acute myeloid leukemia (AML). Methods Pgp negative/positive KB cell lines treated with tumor necrosis factor α/hyperthermia and frozen–thawed AML samples were used as apoptosis/Pgp models. Results For the accurate assessment of apoptosis in samples with unknown Pgp status, it was essential to include a sample with PSC833: in such samples, viable cells always show a Syto16high and apoptotic cells a Syto16low fluorescence. Apoptotic cells loose their Pgp activity early on; in Pgp‐positive cells, the Syto16low apoptotic cells then colocalize with the Syto16low viable cells in the situation minus PSC833. We have developed a gating strategy that, apart from quantifying apoptosis, allowed gating out these apoptotic cells for proper Pgp assessment. By using this strategy, no differences in Pgp activity were found in the treated versus the untreated samples (KB cells: P = 0.779, n = 10; AML cells: P = 0.525, n = 45). Conclusions The use of the combination Syto16/PSC833/7‐AAD provides a sensitive multiparameter flow cytometry method that enables accurate assessment of both apoptosis, cell death, and Pgp function in clinical samples. Cytometry Part B (Clin. Cytometry) 55B:14–21, 2003.

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