Plug flow cytometry extends analytical capabilities in cell adhesion and receptor pharmacology.
BACKGROUND
Plug flow cytometry is a recently developed system for the automated delivery of multiple small boluses or "plugs" of cells or particles to the flow cytometer for analysis. Important system features are that sample plugs are of precisely defined volume and that the sample vessel need not be pressurized. We describe how these features enable direct cell concentration 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 cells expressing the human 7-transmembrane formyl peptide receptor were loaded with the fluorescent probe indo-1 for intracellular ionized calcium determinations. A computer-controlled syringe or peristaltic pump loaded the sample into a sample loop of the plug flow coupler, a reciprocating eight-port valve. When the valve position was switched, the plug of sample in the sample loop was transported to the flow cytometer by a pressure-driven fluid line.


RESULTS
In stirred mixtures of PMNs and CHO cells, we used plug flow cytometry to directly quantify changes in concentrations of nonadherent singlet PMNs. This approach enabled accurate quantification of adherent PMNs in multicell aggregates. We constructed a novel plug flow interface between the flow cytometer and a cone-plate viscometer to enable real-time flow cytometric analysis of cell-cell adhesion under conditions of uniform shear. The High Throughput Pharmacology System (HTPS) is an instrument used for automated programming of complex pharmacological cell treatment protocols. It was interfaced via the plug flow coupling device to enable rapid (< 5 min) flow cytometric characterization of the intracellular calcium dose-response profile of U937 cells to formyl peptide.


CONCLUSIONS
By facilitating the coupling of flow cytometers to other fluidics-based analytical instruments, plug flow cytometry has extended analytical capabilities in cell adhesion and pharmacological characterization of receptor-ligand interactions.
Edwards
B S
BS
Cancer Research and Treatment Center, Department of Pathology, University of New Mexico Health Sciences Center, Albuquerque, New Mexico. 87131, USA. Bedwards@salud.unm.edu
Kuckuck
F W
FW
Prossnitz
E R
ER
Okun
A
A
Ransom
J T
JT
Sklar
L A
LA
D000818:Animals; D001572:Benzofurans; D016466:CHO Cells; D002448:Cell Adhesion / Q000502:physiology*; D006224:Cricetinae; D004867:Equipment Design; D005434:Flow Cytometry / Q000295:instrumentation / Q000379:methods*; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins; D006801:Humans; D007093:Imidazoles; D066298:In Vitro Techniques; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D009504:Neutrophils / Q000502:physiology*; D019007:P-Selectin / Q000032:analysis / Q000235:genetics; D015203:Reproducibility of Results; D013314:Stress, Mechanical; D014162:Transfection; D020298:U937 Cells; D014783:Viscosity
D001572:Benzofurans; D005456:Fluorescent Dyes; D007093:Imidazoles; D008164:Luminescent Proteins; D019007:P-Selectin; D049452:Green Fluorescent Proteins; C082877:fura red