Plug flow cytometry extends analytical capabilities in cell adhesion and receptor pharmacology

Background: Plug flow cytometry is a recently developed system for the auto mated delivery of multiple small boluses or "plugs" of cells or particles t o the flow cytometer for analysis. Important system features are that sampl e plugs are of precisely defined volume and that the sample vessel need not be pressurized. We describe how these features enable direct cell concentr ation determinations and novel ways to integrate flow cytometers with other analytical instruments. Methods: Adhesion assays employed human polymorphonuclear neutrophils (PMNs ) loaded with Fura Red and Chinese hamster ovary (CHO) cells cotransfected with genes for green fluorescent protein (GFP) and human P-selectin. U937 c ells expressing the human 7-transmembrane formyl peptide receptor were load ed with the fluorescent probe indo-1 for intracellular ionized calcium dete rminations. A computer-controlled syringe or peristaltic pump loaded the sa mple into a sample loop of the plug now coupler, a reciprocating eight-port valve. When the valve position was switched, the plug of sample in the sam ple loop was transported to the flow cytometer by a pressure-driven fluid l ine. Results: In stirred mixtures of PMNs and CHO cells, we used plug flow cytom etry to directly quantify changes in concentrations of nonadherent singlet PMNs. This approach enabled accurate quantification of adherent PMNs in mul ticell aggregates. We constructed a novel plug flow interface between the f low cytometer and a cone-plate viscometer to enable real-time flow cytometr ic analysis of cell-cell adhesion under conditions of uniform shear. The Hi gh Throughput Pharmacology System (HTPS) is an instrument used for automate d programming of complex pharmacological cell treatment protocols. It was i nterfaced via the plug flow coupling device to enable rapid (<5 min) flow c ytometric characterization of the intracellular calcium dose-response profi le of U937 cells to formyl peptide. Conclusions: By facilitating the coupling of flow cytometers to other fluid ics-based analytical instruments, plug flow cytometry has extended analytic al capabilities in cell adhesion and pharmacological characterization of re ceptor-ligand interactions.