ABSTRACT
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.
Subject(s)
Flow Cytometry/instrumentation , Flow Cytometry/methods , Immunophenotyping/instrumentation , Immunophenotyping/methods , Lasers , Membrane Glycoproteins , Animals , Antibodies, Monoclonal , Antigens, CD/analysis , Antigens, CD/immunology , B-Lymphocytes/immunology , CD24 Antigen , CD3 Complex/analysis , CD3 Complex/immunology , CD5 Antigens/analysis , CD5 Antigens/immunology , Color , Fluorescein-5-isothiocyanate , Fluorescent Dyes , Leukosialin , Mice , Mice, Inbred BALB C , Optics and Photonics , Receptors, Complement 3d/analysis , Receptors, Complement 3d/immunology , Sialoglycoproteins/analysis , Sialoglycoproteins/immunology , Spleen/cytologyABSTRACT
Calcium flux measurements of different subpopulations of cells by flow cytometry are important in understanding complex interactions in the immune system. This paper discusses the use of the difference of Log signals as a preferred method for obtaining this information simultaneously with other immunofluorescence parameters. We describe simple modifications to a commercial instrument that enables the measurement of calcium flux in addition to three immunofluorescence parameters. Finally, we show an application of this technique to measuring calcium flux of T cell subsets in human blood. We show that different subsets of peripheral CD4 T cells have significantly different capabilities to flux calcium after CD3 stimulation. These differences are related to the functional capacities of the cells within these subsets.
Subject(s)
Antigens, CD/analysis , Calcium/metabolism , Flow Cytometry/instrumentation , Flow Cytometry/methods , T-Lymphocyte Subsets/cytology , T-Lymphocyte Subsets/metabolism , CD4 Antigens/analysis , Cell Line , Electronics , Fluorescent Dyes , Humans , Indoles , Leukocyte Common Antigens/analysis , Reproducibility of Results , Sensitivity and Specificity , T-Lymphocyte Subsets/immunology , Tumor Cells, CulturedABSTRACT
Current cell sorting machines do not preserve the individual identity of processed cells; after analysis, the cells are assigned to a subpopulation where they are pooled with other similar cells. This paper reports progress on a system that sorts cells individually to precise locations on a microscope slide and preserves them for further observation with a light microscope while recording flow measurement data for each cell. Various electronic and mechanical modifications to an existing sorting machine are described that increase drop placement accuracy and permit individual cell sorting.
Subject(s)
Cytological Techniques , Lymphocytes/cytology , Animals , Cell Separation , Cytological Techniques/instrumentation , Humans , Mice , Microscopy, Fluorescence , Microspheres , Models, BiologicalABSTRACT
Flattened cells, such as red blood cells, epithelial cells, and sperm of many species, cause problems for fluorescence-activated cell analysis and sorting machines because the flow systems of such devices are unable to control the orientation of these cells as they flow past the detectors. For this reason, the fluorescence or scattered light measurements for identical cells may vary greatly. A flow geometry is here described that orients flat cells in a coaxial flow system so that each cell presents the same aspect to the observation device. A wedge-shaped exit on the sample injection tube in a coaxial flow system is sufficient to produce the desired orientation effect when used with low sample flow rates. Data is presented showing the effect of orientation of fixed chicken erythrocytes on histograms of small forward-angle light-scattering measurements.
Subject(s)
Cytological Techniques , Fluorescence , Light , Mathematics , Rheology , Scattering, RadiationABSTRACT
Experimental observations of the breakup of a liquid jet issuing from a vibrated nozzle support the view that particles passing through the orifice tend to create a surface disturbance which, depending on the phase relative to the nozzle vibration, may either add to or subtract from the disturbance created by the nozzle vibration. The result may be either a shortening or lengthening of the jet breakoff length, depending on phase. The implications for sorting large cells with a fluorescence-activated cell sorter are discussed.
