Mixing small volumes for continuous high‐throughput flow cytometry: Performance of a mixing Y and peristaltic sample delivery

Background Online mixing for continuous high‐throughput flow cytometry has not been previously described. A simple, general high‐throughput method for mixing and delivery of submicroliter volumes in laminar flow at low Reynolds numbers would be widely useful. Materials and Methods We describe a micromixing approach that is compatible with commercial autosamplers, flow cytometry, and other detection schemes that require mixing of components that have been introduced into laminar flow. The scheme is based on a previous approach to high‐throughput flow cytometry (HyperCyt™, Kuckuck et al.: Cytometry 44:83–90, 2001). We showed that samples from multiwell plates that have been picked up by an autosampler can be separated during delivery by the small air bubbles introduced during the transit of the autosampler probe from well to well. Here, a particle sample flowing continuously is brought together in a Y with reagent samples from wells, which have been separated by bubbles. Results In the effluent stream, the particles and reagents are mixed, most likely as a result of peristaltic action, and reagents from individual wells can be resolved. The sample volumes that can be mixed with this technology are submicroliter in volume, and samples can be mixed at rates up to at least 100/samples per minute. With the current device, carryover between samples can be eliminated if the mixing system is flushed with several volumes of buffer. The anticipated throughput for screening is expected to be at least 20 samples per minute. Conclusions The high‐throughput approach and peristaltic mixing in HyperCyt™ serve to integrate autosamplers with submicroliter detection volumes for analysis in flow cytometry or in microfluidic channels. Cytometry 47:183–191, 2002. © 2002 Wiley‐Liss, Inc.