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Sims, C. , Meredith, G. , Krasieva, T. , Berns, M. , Tromberg, B. , and Allbritton, N. L. (1998) Laser-micropipet combination for single-cell analysis, Anal Chem 70, 4570–4577. 5. , and Krylov, S. N. (2003) Use of capillary electrophoresis and endogenous fluorescent substrate to monitor intracellular activation of protein kinase A, Anal Chem 75, 3720–3724. 6. Brown, R. , and Audet, J. (2007) Sampling efficiency of a single-cell capillary electrophoresis system, Cytometry A 71, 882–888. 7. Brown, R.

10. DAPI: 4¢-6-diamidino-2-phenylindole (Invitrogen), diluted to 300 nM in PBS. 11. Trypan blue (Sigma) exclusion method, diluted 1:5 in deionized water (see Note 1). 12. 5 mM (Invitrogen). 3. Single-Cell Seeding 1. Cell scraper (BD Falcon). 2. Flow cytometer with sorting capabilities. ). For more information on cell sorting (into plates), contact your instrument supplier for help. This protocol describes the procedure with a FACS Vantage SE Cell Sorter fitted with a 100 mm nozzle, a plate holder for the motorized x/y stage, a slide holder to be placed onto the plate holder, and the software CloneCyt Plus (all from BD Biosciences) (see Note 2).

Wall structures. Wells on a plate enable parallel analysis of multiple samples and have long been applied in life science. Depending on the application, different modifications have been performed. For example, standard 96-well plates with different bottom materials have been developed for improved transparency and reduced background flourescence, when detecting cell growth using a microscope. By miniaturizing, more samples (wells) can be run in parallel leading to a higher throughput. Microwells can in many cases be considered a simple and straightforward technique, in terms of theory, practical handling and fabrication.

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