Biomedical Engineering Reference
In-Depth Information
temperature fluctuations, cell density, timing of reagent addition, and for antibody-
based assays, staining volume, time, and temperature. In addition, when acquiring
samples on the flow cytometer or any analytical instrument for that matter, changes in
detection sensitivity, optics, or flow rates may affect results. With the FCB method,
many of these variables are minimized. By staining many samples together, one
eliminates staining variability. In addition, because positive and negative controls can
be stained with drug-treated samples, complex normalization procedures are not
needed. Each barcoded sample can be normalized to itself. Another advantage is that
data on many samples are acquired simultaneously on the instrument. Therefore, if
there is a mechanical error, all the samples in that barcoded sample will be affected
equally. If the samples had been run in sequence, only some wells would have been
affected, leading to a large drift in data. Thus, FCB eliminates variability on the
instrument acquisition end, leaving the major sources of variability at the front end of
the experiment, with cell preparation, distribution, drug treatment, and reagent
addition. In our studies, FCB assays are superior to singly stained assays with Z
0
values of 0.8 or greater in most cases [11].
Increased Acquisition Speed In typical cytometers, the largest bottleneck in
acquisition rate is the switching of samples from one tube to the next. Because
FCB combines multiple samples into one, no switching of samples is required.
Even without plate loading hardware, one can easily run the equivalent of a 96-well
plate in less than 5min using FCB, because the acquisition is from one continuous
tube. Therefore, on any standard cytometer setup, a researcher can load and acquire
10 or more 96-well plates worth of samples in 1 h, dramatically improving
throughput.
15.4.2 High-Throughput Autosamplers
In addition to the FCB assay, recent advances in hardware have made it possible to
analyze a 384-well plate in less than 15min. Therefore, screening 10,000 wells in
one day is entirely feasible, eliminating sample throughput as a major concern in
flow cytometry. This platform, called plug flow cytometry, uses a HPLC-style
autosampler and peristaltic pump to serially sample from the assay plate (for review,
see Refs [12-14]). Each sample is separated by a small air gap, or plug, so that the
samplesdonotmixastheytraveluptheautosampler tubing and into the cytometer.
This was a counterintuitive innovation because most researchers are taught to never
introduce air bubbles into the flow cell of the flow cytometer. However, because
most of the fluid entering the flow cell at any given time is sheath fluid, the small air
bubbles that are introduced in the sample line do not cause any major perturbations.
The plug flow cytometry platform has been used extensively for drug screening as
part of the NIH Roadmap initiative. Millions of wells have been screened in assays
on beads, in yeast cells, and mammalian cells. These include receptor binding
assays, enzyme modifications, proteases, GTPases, and GPCRs [15]. This work is
pioneering in its application of flow cytometry to high-throughput, high-content
screening.
Search WWH ::
Custom Search