title: Flow cytometry APC-tandem dyes are degraded through a cell-dependent mechanism.
abstract: Technological developments of multiparametric flow cytometry come along with the generation of new dyes. The APC-tandem dyes, which combine the fluorophores APC and Cy7/H7, allow the detection of a specific signal in the APC-Cy7/H7 channel along with an unexpected nonspecific signal in the APC channel. Depending on the magnitude of the latter, it may be a handicap for interpreting the data of multicolor labeling experiments. We investigated the alteration of the APC-tandem dyes by labeling peripheral blood cells with antibodies directed toward leukocyte surface proteins and by analyzing cells by flow cytometry. Our results show that the APC-Cy7/H7 tandem fluorochromes degraded over time. Nonspecific APC signal was observed with the various antibodies tested only upon cell attachment but not under bead linkage. Moreover, the percentage of degradation of the APC-Cy7/H7 dyes was dependent on the cell type analyzed. Interestingly, nonspecific APC signal strongly decreased when the metabolic activity of immunolabeled cells was inhibited or when cells were incubated with vitamin C. This study demonstrates that the APC-tandem dyes are the target of cell-dependent degradation, which may be antagonized. These findings will allow cytometer users to optimize their multicolor panels.
authors.affiliation: UMR U978 INSERM-Université Paris 13, UFR Santé Médecine et Biologie Humaine, Bobigny, France.
authors.forename: Christine
authors.initials: C
authors.lastname: Le Roy
authors.forename: Nadine
authors.initials: N
authors.lastname: Varin-Blank
authors.forename: Florence
authors.initials: F
authors.lastname: Ajchenbaum-Cymbalista
authors.forename: Rémi
authors.initials: R
authors.lastname: Letestu
mesh_terms: D000906:Antibodies / Q000378:metabolism; D001205:Ascorbic Acid / Q000494:pharmacology; D052160:Benzothiazoles; D001773:Blood Cells / Q000187:drug effects / Q000378:metabolism* / Q000528:radiation effects; D002232:Carbocyanines; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D017493:Leukocyte Common Antigens / Q000378:metabolism; D008027:Light; D010798:Phycocyanin / Q000378:metabolism*; D019810:Sodium Azide / Q000494:pharmacology; D013194:Staining and Labeling; D013696:Temperature; D013997:Time Factors; D016707:Tissue Fixation
chemical_list: D000906:Antibodies; D052160:Benzothiazoles; D002232:Carbocyanines; D005456:Fluorescent Dyes; C002497:allophycocyanin; D010798:Phycocyanin; C011204:cyanine dye 7; D019810:Sodium Azide; D006861:Hydrogen Peroxide; D017493:Leukocyte Common Antigens; D001205:Ascorbic Acid