BACKGROUND: This study describes a three laser flow cytometer, reagents, and software used to simultaneously evaluate nine distinct fluorescent parameters on one cell sample. We compare the quality of data obtained with (1) full software compensation and (2) the use of partial spectral compensation of selected pairs of parameters in analog hardware, in combination with final software compensation. An application characterizing low frequency murine B cell subpopulations is given. METHODS: The fluorochromes used are: fluorescein (FITC), phycoerythrin (PE), Cy5PE and Cy7PE, excited at 488 nm by an argon laser; Texas Red (TR), allophycocyanin (APC), and Cy7APC excited at 595 nm by a pumped dye laser; and cascade blue (CB) and cascade yellow (CY) excited at 407 nm by a violet-enhanced krypton laser. Custom additions to commercial electronics and an extended optical bench allow the measurement of these nine parameters plus forward and side scatter light signals. RESULTS: We find the use of partial analog compensation reduces the variation in the background staining levels introduced by the compensation process. Novel B cell populations with frequencies below 1% are characterized. CONCLUSIONS: Nine color flow cytometry is capable of providing measurements with high information content. The choice of reagent-dye combinations and the ability to compensate in multi-parameter measurement space are crucial to obtaining satisfactory results.
BACKGROUND: This study describes a three laser flow cytometer, reagents, and software used to simultaneously evaluate nine distinct fluorescent parameters on one cell sample. We compare the quality of data obtained with (1) full software compensation and (2) the use of partial spectral compensation of selected pairs of parameters in analog hardware, in combination with final software compensation. An application characterizing low frequency murine B cell subpopulations is given. METHODS: The fluorochromes used are: fluorescein (FITC), phycoerythrin (PE), Cy5PE and Cy7PE, excited at 488 nm by an argon laser; Texas Red (TR), allophycocyanin (APC), and Cy7APC excited at 595 nm by a pumped dye laser; and cascade blue (CB) and cascade yellow (CY) excited at 407 nm by a violet-enhanced krypton laser. Custom additions to commercial electronics and an extended optical bench allow the measurement of these nine parameters plus forward and side scatter light signals. RESULTS: We find the use of partial analog compensation reduces the variation in the background staining levels introduced by the compensation process. Novel B cell populations with frequencies below 1% are characterized. CONCLUSIONS: Nine color flow cytometry is capable of providing measurements with high information content. The choice of reagent-dye combinations and the ability to compensate in multi-parameter measurement space are crucial to obtaining satisfactory results.
Authors: Li-Sheng Lu; James Tung; Nicole Baumgarth; Ometa Herman; Michael Gleimer; Leonard A Herzenberg; Leonore A Herzenberg Journal: Proc Natl Acad Sci U S A Date: 2002-02-26 Impact factor: 11.205
Authors: Mark M Hammer; Nikesh Kotecha; Jonathan M Irish; Garry P Nolan; Peter O Krutzik Journal: Assay Drug Dev Technol Date: 2009-02 Impact factor: 1.738
Authors: Bridget E McLaughlin; Nicole Baumgarth; Martin Bigos; Mario Roederer; Stephen C De Rosa; John D Altman; Douglas F Nixon; Janet Ottinger; Judy Li; Laurel Beckett; Barbara L Shacklett; Thomas G Evans; David M Asmuth Journal: Cytometry A Date: 2008-05 Impact factor: 4.714
Authors: Bridget E McLaughlin; Nicole Baumgarth; Martin Bigos; Mario Roederer; Stephen C De Rosa; John D Altman; Douglas F Nixon; Janet Ottinger; Carol Oxford; Thomas G Evans; David M Asmuth Journal: Cytometry A Date: 2008-05 Impact factor: 4.714