Dead cell discrimination with 7-amino-actinomycin D in combination with dual color immunofluorescence in single laser flow cytometry.

@article{citeulike:3000377, abstract = {Identification of nonviable cells in immunofluorescently stained cell populations is essential for obtaining accurate data. Fluorescent non-vital DNA dyes, particularly propidium iodide (PI), have been used routinely in flow cytometry for discrimination of dead cells from viable cells on the basis of fluorescence. We describe here the use of an alternative DNA dye, 7-amino-actinomycin D (7-AAD), which can replace PI for the exclusion of nonviable cells. As an example, we present in this paper the utilization of 7-AAD on various leukemic cell lines for dead cell exclusion whenever the viable cell population could not be discriminated reliably from nonviable cells on the light scatter histogram; 7-AAD is suitable for dead cell discrimination in lengthy experiments because it is efficiently excluded by intact cells and has a high DNA binding constant. In addition, the dye is valuable in combination with phycoerythrin (PE)-fluorescence dual-color flow cytometry on a single argon laser instrument, since its emission in the far red can easily be separated from the emission of PE; 7-AAD was used on fluoresceinisothiocyanate (FITC) and PE surface-labeled human thymocytes for characterization of the dying subpopulation of cells which is undergoing programmed cell death. In this heterogeneous cell preparation, the spectral properties of the dye permitted the classification of viable and nonviable cell subpopulations by multiparameter analysis.}, address = {Department of Medicine, UCLA School of Medicine 90024.}, author = {Schmid, I. and Krall, W. J. and Uittenbogaart, C. H. and Braun, J. and Giorgi, J. V. }, citeulike-article-id = {3000377}, comment = {PI, Invitrogen PNN1011 (50 ug/mL, 2 mL) - use at 1 ug/mL final,ie, 2 uL per 100 uL cells (10^6). 7AAD, BD 559925 (50 ug/mL, 2 mL) - use 5 uL (0.25 ug) per 10^6 cells [typically in 100-200 uL] ... incu 5-10 min on ice before read.. 7-AAD = FL3 (FACScan or FACS Calibur) compatible with FITC, PE, APC... em. 630/30 PI = FL3, em. 630/22. PI also in PE channel when using FACScan (?) 7-AAD preferred marker (1 ug/ml final conc) becoz of less spectral overlap of PE and 7-AAD than PE and PI. However, 7-AAD is not as bright as PI. If care is observed when setting up the parameters on the cytometer, FITC, PE and PI can be combined effectively. }, doi = {10.1002/cyto.990130216}, issn = {0196-4763}, journal = {Cytometry}, keywords = {apoptosis, facs, fluorophore, method}, number = {2}, pages = {204--208}, posted-at = {2008-07-15 00:36:42}, priority = {2}, title = {Dead cell discrimination with 7-amino-actinomycin D in combination with dual color immunofluorescence in single laser flow cytometry.}, url = {http://dx.doi.org/10.1002/cyto.990130216}, volume = {13}, year = {1992} }

TY - JOUR ID - citeulike:3000377 L3 - citeulike-article-id:3000377 TI - Dead cell discrimination with 7-amino-actinomycin D in combination with dual color immunofluorescence in single laser flow cytometry. JF - Cytometry VL - 13 IS - 2 SP - 204 EP - 208 AD - Department of Medicine, UCLA School of Medicine 90024. SN - 0196-4763 N2 - Identification of nonviable cells in immunofluorescently stained cell populations is essential for obtaining accurate data. Fluorescent non-vital DNA dyes, particularly propidium iodide (PI), have been used routinely in flow cytometry for discrimination of dead cells from viable cells on the basis of fluorescence. We describe here the use of an alternative DNA dye, 7-amino-actinomycin D (7-AAD), which can replace PI for the exclusion of nonviable cells. As an example, we present in this paper the utilization of 7-AAD on various leukemic cell lines for dead cell exclusion whenever the viable cell population could not be discriminated reliably from nonviable cells on the light scatter histogram; 7-AAD is suitable for dead cell discrimination in lengthy experiments because it is efficiently excluded by intact cells and has a high DNA binding constant. In addition, the dye is valuable in combination with phycoerythrin (PE)-fluorescence dual-color flow cytometry on a single argon laser instrument, since its emission in the far red can easily be separated from the emission of PE; 7-AAD was used on fluoresceinisothiocyanate (FITC) and PE surface-labeled human thymocytes for characterization of the dying subpopulation of cells which is undergoing programmed cell death. In this heterogeneous cell preparation, the spectral properties of the dye permitted the classification of viable and nonviable cell subpopulations by multiparameter analysis. KW - apoptosis KW - facs KW - fluorophore KW - method N1 - PI, Invitrogen PNN1011 (50 ug/mL, 2 mL) - use at 1 ug/mL final,ie, 2 uL per 100 uL cells (10^6). 7AAD, BD 559925 (50 ug/mL, 2 mL) - use 5 uL (0.25 ug) per 10^6 cells [typically in 100-200 uL] ... incu 5-10 min on ice before read.. 7-AAD = FL3 (FACScan or FACS Calibur) compatible with FITC, PE, APC... em. 630/30 PI = FL3, em. 630/22. PI also in PE channel when using FACScan (?) 7-AAD preferred marker (1 ug/ml final conc) becoz of less spectral overlap of PE and 7-AAD than PE and PI. However, 7-AAD is not as bright as PI. If care is observed when setting up the parameters on the cytometer, FITC, PE and PI can be combined effectively. AU - Schmid, I AU - Krall, WJ AU - Uittenbogaart, CH AU - Braun, J AU - Giorgi, JV PY - 1992/// UR - http://dx.doi.org/10.1002/cyto.990130216 ER -