54
appeared within the first order of the logarithmic scale of
fluorescence.- Analyse the remaining stained samples at the settings defined
in step 4. - Collect 10,000 events at a set standard ‘Low’ event rate.
- Analyse the acquired data to create one-parameter fluorescence
histogram overlays and two-parameter dot plots (see Note 7) [ 8 ]. - For all fluorescence dot plots, gate around the un-stained con-
trol in relation to the CV-AM fluorescence and gate around
the stained live/zero drug control in relation to the SG fluo-
rescence (see Notes 7 and 8 ). - Obtain percentages of the total cell population residing in each
gate for each parameter or each time-point (see Notes 1 and
7 – 9 ). - The data obtained can then be plotted as a percentage of the
total population over the time-course of the experiment. Two
types of graphs can be plotted either comparing different antibi-
otic concentrations for the same population gate or comparing
different population gates for the same antibiotic concentration
(see Note 9). - Differences between antibiotic treatments can be analysed
using two-way ANOVA with appropriate post-hoc tests. - Inter-experiment variability can be assessed by performing a
coefficient of variance test on the percentage values of the total
population in each gate across three independent experiments.
4 Notes
- The authors use Calcein violet-AM (CV-AM), cat no. C34858,
and SYTOX-green (SG), Cat no. S7020 (Invitrogen, Life
Technologies), which have been found to be the optimal
reagents for success in this method. DMSO is used from indi-
vidual 5 mL vials Cat no. D2650-5x5ML. A fresh vial should
be opened each time the assay is performed. - The published method [ 2 ] used a CyAn ADP (9 colour)
Analyser (Beckman Coulter) with attached Cytek plate loader
as the capabilities of this flow cytometer possess the required
specifications for these experiments, in particular 405 nm and
488 nm lasers, good resolution for detecting bacteria, and
530/40 & 450/50 BP filters. - The published method [ 2 ] used software package, Sigmaplot,
for graphical and statistical analyses. However, other software
packages, e.g., Graphpad Prism (version 6), may be used
according to individual preference.
3.4 Data Analysis
Charlotte L. Hendon-Dunn et al.