INTRODUCTION TO FLOW CYTOMETRY - Flow Cytometry in Drug Discovery and Development - page 13

(Figure 1.7b and c). Although hydrodynamic focusing streams the cells into a single
file, occasionally two cells stick together. A doublet made of two single positive cells
(eachonepositivefor adifferent fluorochrome) canbeerroneouslyrecordedas adouble
positive cell. Hence, doublet discrimination is crucial. Doubletswill have a higher FSC
height/FSC area ratio as compared to singlets. One can set a gate around the events in
which there is a linear relationship between FSC-Hand FSC-A (Figure 1.7d) and these
cells can then be analyzed for different markers (Figure 1.7e-h).
1.8.3 Two-Color Dot Plot Versus Contour Plot
The fluorescence of two differentmarkers can be represented in2Dusing the two-color
dot plot (Figure1.8a-d) or acontour plot (Figure1.8b 0 -d 0 ).Thereareadvantages todata
displayed in eithermode and data are amenable to either type of analysis. In Figure 1.8,
identical data are displayed as a dot plot and a contour plot. In a dot plot, each dot
represents one or more events (events are usually cells that have passed the criteria of
thresholding and gating) that are determined by the user. The density of events can be
color coded (e.g., red implying highest density). The events are gated on the lymphoid
gate on the basis of FSC and SSC (Figure 1.8a) with a second gate to include only
lymphoid cells that are CD3 positive (Figure 1.8c 0 ). There exists a small population of
cells positive for both CD4 and CD8, which is visible in the dot plot (Figure 1.8d).
Contour plots show the same data inwhich rings represent a defined percentage of total
eventswith a particular combination of fluorescence intensities (Figure 1.8b 0 -d 0 ). This
type of data display removes the outliers allowing one to clearly see subpopulations of
cells. In addition, one can smoothen the contour plots making it even more difficult to
visualize the rarepopulation (Figure 1.8d 0 , CD4 þ /CD8 þ cells).Using contour plots as
a formattingoption, otherdisplayoptions include linear densityand logdensitycontour
plots in which the contour lines are defined as a percentage of the maximum numbers.
There is an additional option in which one can combine a contour plot and a dot plot in
which dots are displayed below the lowest contour line allowing the observation of the
rare population in a contour plot. Despite different display options, the data quadrant
statistics would be identical in both the dot plot and the contour plot.
1.9 SORTING
Cells (or particles) of interest (expression of desired markers) can be purified or
sorted [71]. In most flow cytometers equipped with sorting capabilities, the liquid
sheath stream is regularly broken into droplets by the vibration of the piezoelectric
crystal attached to the flow chamber. A cell passing through the laser meeting the
selectioncriteriabasedon thefluorescencepattern is electricallycharged in thedroplet.
These droplets containing the charged cells are then deflected and collected into
awaiting tubes/wells or onto slides. Depending on the further downstreamapplications
of these sortedcells, theymayhave tobecollected inappropriatebuffersor under sterile
conditions. The temperatureof the sheathfluidand sample collection tubesmayneed to
be controlled. In the example shown, mouse spleen cells to be sorted are shown on the