Biomedical Engineering Reference
In-Depth Information
gradient of soluble compound in
2min. The plug flow technology led to funding of a
BRP from NIH to develop the current generation of HT flow cytometry. HyperCyt
uses a peristaltic pump in combination with an autosampler to boost assay throughput.
As the sampling probe of the autosampler moves from well to well, a peristaltic pump
sequentially aspirates particle suspensions from each well. Between wells, the running
pump draws a bubble of air into the sample line resulting in the delivery of a series of
bubble-separated samples [32]. Data from microplate wells are collected in a single
data file. The time-resolved data, with periodic gaps corresponding to the passage of the
sample-separating air bubbles, are analyzed by software. 384-Well plates with multi-
plexed data sets are routinely sampled in 10min with sample volumes of 1-2
Ls. We
have adapted the particle counting ability of flow cytometry for HT analysis of
compound solubility.
These developmental activities ultimately led to NIH Roadmap funding from the
Molecular Libraries Initiative. As a comprehensive screening center in the pilot phase,
the New Mexico Molecular Libraries Screening Center developed capabilities in
target development, HTS, as well as cheminformatics, and synthetic chemistry for
optimization of biological activity of leads identified through the screening process. In
the production phase, our name changed to UNMCMD to reflect our status as a
specialty probe production center focusing on HTS flow cytometry. The screens are
intended to identify active molecules, and to chemically optimize those molecules for
biological activity. HT flow cytometry tools enable high-throughput sample handling,
high content analysis, and real-time measurements of cell response. The HyperCyt
platform delivers microliter-sized samples from 384-multiwell plates at typical
sampling rates of 40 wells per minute for end point assays with multiplexing of six
or more targets simultaneously. Our experience indicates that virtually any molecular
assembly or cell response can be displayed in a format compatible with flow
cytometry. Moreover, by creating a suspension array of particles, assays and
responses can be highly multiplexed or performed on complex cell populations
without compromising throughput.
m
4.4 PLATE-BASED FLOW CYTOMETRY
During 2000 and 2010, plate-based cytometry has become available through most
manufacturers of flow cytometers (Accuri, Beckman Coulter, Becton-Dickinson,
Guava, and Luminex) as well as flow cytometry device companies (e.g., Cytek). Since
flowcytometersarepressurized, themajorityof theseplate-basedsystemsdependupon
two common principles: (1) samples from individual wells are injected into the flow
cytometer one well at a time; and (2) the contents of each well are treated as a single
FCS file. Inmost cases, the contents of individual wells have been drawn into a syringe
prior to injection; in some cases, the wells have been sealed and pressurized (Cytek)
for sample delivery; and recently in one case the contents of a well have been sipped
by the flow cytometer (Accuri), where the plate has been repositioned for each well.
HTS has been typically defined as screening rates of 100,000 samples or more per
day. Systems that create an FCS file from each well have been reported at rates of
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