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The histogram graph has ﬂuorescent intensity on the x-axis with the number of cells in each channel on the y-axis. 1). A histogram can only represent the intensity of a single parameter (scatter or ﬂuorescence). Dot plots (bivariate display, two parameter histograms, scattergram, bitmap) unlike histograms show the correlated distribution for two parameters, such as forward scatter versus side scatter, with each cell being represented as a dot, positioned on the x and y axes according to the intensities detected for that cell.
2. Upper-right quadrant represents cells dual-positive for the descriptors on both the x- and y-axis. 3. Upper-left quadrant represents cells positive for the y-axis descriptor, but negative for the x-axis descriptor. 4. Lower-right quadrant represents cells positive for the x-axis descriptor, but negative for the y-axis descriptor. PRINCIPLES OF FLOW CYTOMETRY SAMPLE PREPARATION The aim of sample preparation is to produce a monodisperse suspension; this is a suspension of single cells with minimal aggregation.
For instance, enzymatic preparative techniques can alter cell-surface antigens and affect cell viability. Once a monodisperse suspension is gained the cells are labelled by incubation with a ﬂuorescent tag under appropriate conditions. This may be a ﬂuorescent dye, ﬂuorescent conjugated antibody or ligand, which is speciﬁc for the “antigen” to be measured. The ﬂuorescent probes and monoclonal antibodies may bind nonspeciﬁcally and it is essential that care is taken to minimize the chances of cross-reactions.