Nine color eleven parameter immunophenotyping using three laser flow cytometry.
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.
Bigos
M
M
Department of Genetics, Stanford University School of Medicine, California 94305-5318, USA. bigos@stanford.edu
Baumgarth
N
N
Jager
G C
GC
Herman
O C
OC
Nozaki
T
T
Stovel
R T
RT
Parks
D R
DR
Herzenberg
L A
LA
D000818:Animals; D000911:Antibodies, Monoclonal; D015703:Antigens, CD / Q000032:analysis / Q000276:immunology; D001402:B-Lymphocytes / Q000276:immunology; D051927:CD24 Antigen; D017252:CD3 Complex / Q000032:analysis / Q000276:immunology; D018956:CD5 Antigens / Q000032:analysis / Q000276:immunology; D003116:Color; D005434:Flow Cytometry / Q000295:instrumentation* / Q000379:methods*; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D016130:Immunophenotyping / Q000295:instrumentation* / Q000379:methods*; D007834:Lasers; D051917:Leukosialin; D008562:Membrane Glycoproteins; D051379:Mice; D008807:Mice, Inbred BALB C; D055095:Optics and Photonics; D017464:Receptors, Complement 3d / Q000032:analysis / Q000276:immunology; D012795:Sialoglycoproteins / Q000032:analysis / Q000276:immunology; D013154:Spleen / Q000166:cytology
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D051927:CD24 Antigen; D017252:CD3 Complex; D018956:CD5 Antigens; C497890:Cd24a protein, mouse; D005456:Fluorescent Dyes; D051917:Leukosialin; D008562:Membrane Glycoproteins; D017464:Receptors, Complement 3d; D012795:Sialoglycoproteins; C497863:Spn protein, mouse; D016650:Fluorescein-5-isothiocyanate