Fast kinetics

John P. Nolan (
Sun, 9 Mar 97 16:12:34 MST

At the NFCR, Larry Sklar and I have spent a lot of time making kinetic
measurements using flow cytometry. To measure the response of a cell to a
stimulus, in addition to rapidly delivering sample to the cytometer, one
must also demonstrate the reagents were combined quantitatively (so you know
the concentrations of all species) and mixed completely.

With respect to rapid kinetics, the following generalization can be made: If
you use a stir bar to mix reagents, you will never get the dead-time below
several seconds. Which is not to say you can not measure kinetics at ealier
times, it is just that the kinetics you measure will be a combination of
mixing and reaction kinetics.

The approach we have found most useful is adapted from stopped flow kinetic
measurements in enzymology, for example, where reagents are mixed in a
mixing tee. Syringe-based sample delivery systems enable quantitative
reagent proportioning, mixing, and delivery in under half a second
(Cytometry 21:223-9). In principle, flow injection technology should allow
similar performance. Setting up such a system is not a trivial undertaking
(in terms of money or time), but if one has a commitment to making kinetic
measurments it is well worth it. Alternately, the Rapid Kinetic Flow
Cytometer at the NFCR is available for use in collaborative projects.
Proposals for its use may be sent to me or Larry Sklar.

John Nolan
John P. Nolan, PhD
National Flow Cytometry Resource
Life Sceinces Division, LS-5, M-888
Los Alamos National Laboratory
Los Alamos, NM 87545
505-665-3024 FAX

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