Novel multiparameter flow cytometry assay using Syto16 for the simultaneous detection of early apoptosis and apoptosis-corrected P-glycoprotein function in clinical samples.
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 alpha/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 Syto16(high) and apoptotic cells a Syto16(low) fluorescence. Apoptotic cells loose their Pgp activity early on; in Pgp-positive cells, the Syto16(low) apoptotic cells then colocalize with the Syto16(low) 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.
van der Pol
Marjolein A
MA
Department of Hematology VU University Medical Center, Amsterdam, The Netherlands.
Broxterman
Henk J
HJ
Westra
Guus
G
Ossenkoppele
Gert J
GJ
Schuurhuis
Gerrit J
GJ
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism*; D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis / Q000502:physiology*; D045744:Cell Line, Tumor; D005434:Flow Cytometry / Q000379:methods*; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000187:drug effects; D006801:Humans; D007951:Leukemia, Myeloid / Q000378:metabolism* / Q000473:pathology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D017304:Annexin A5; D005456:Fluorescent Dyes; D014409:Tumor Necrosis Factor-alpha