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Acknowledgments
Funding was received from Department of Health Grant in Aid and
the National Institute of Health Research. The views expressed in
this publication are those of the authors and not necessarily those of
Public Health England, the National Institute for Health Research,Fig. 2 Worked example of flow cytometry data capture and analysis. A single cell population is selected by
placing an elliptical gate on the forward scatter vs. side scatter dot plot. This gate is applied to a FL1 vs FL6
dot-plot. Controls to aid with the subsequent gating strategy include heat-killed bacteria that are either un-
stained, stained with SG, or dual stained with CV-AM and SG. Polygonal population gates are placed according
to the position of the populations of cells from the various controls. These controls enable the placement of
gate 4, and verification that there is no spill-over of fluorescence from SG staining into the FL6 channel, which
was used to measure CV-AM fluorescence intensity. Controls containing zero levels of antibiotic are stained
with CV-AM only, as well as dual-stained; this enables the placement of population gate 2 and verified that
there is no spill-over of fluorescence from CV-AM staining into the FL1 channel, which is used to measure SG
fluorescence intensity. Percentage proportions of the total cell population within each gate are obtained and
can be presented graphically
Charlotte L. Hendon-Dunn et al.