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Biomedical subjects

C H Pollema

Publications and source records attributed to C H Pollema.

7 recordsLinked to original sources
PubMed1995

Configurations of a flow injection system for perfusion studies of adherent cells.

This paper describes three configurations of a flow injection apparatus designed to be the fluidic drive for perfusion studies of cultured adherent cells. The apparatus was coupled to a flow-through perfusion chamber that was specifically designed for live cell perfusion using fluorescence microscopy as the detector. The instrument consists of two linear syringe pumps and a multiposition selector valve which, under computer control, allowed sequential injection, fluid switching, and flow injection to be performed with minimal system reconfiguration. When the apparatus is coupled to a dual inlet perfusion chamber, target cells can be exposed to very steep reagent pulses, while the traditional single inlet perfusion chamber allows more flexibility and provides a more gradual increase in reagent concentration. The most significant salient feature of the system is the ability to generate very steep pulses--a desirable feature for cell perfusion studies.

Cell Adhesion↗
PubMed1995

Flow injection fluorescence microscopy applied to a rapid cell surface immunoassay.

A perfusion system for fluorescence microscopy that utilized a flow injection system was developed and used to study cell surface antibody binding on viable cells grown in monolayer cultures on coverslips. A polyclonal cell-specific antiserum used to probe the cell surface was monitored by indirect immunofluorescence. The flow injection system was completely automatic and allowed controlled perfusion of cell surfaces with the desired sequence of antibodies. The individual steps of the indirect assay were studied to determine the binding behavior of the primary cell surface antibody, the labeled second antibody, and control of nonspecific binding. Under stopped flow conditions, the second antibody was maximally bound within 10 min, while constant mixing of the second antibody solution over the cells resulted in maximal binding within as little as 6 min. A primary antibody contact time of 2 min followed by a wash and then exposure to the second antibody for 2 min showed that this fast automated procedure could distinguish specific from nonspecific binding of cell surface antibodies to the same set of cells for several repeated exposures. The flow injection fluorescence microscopy system can be automated to allow screening for cell surface antibodies and to study their interaction with specific cell surface antigens.

Animals↗
PubMed1994

Flow injection renewable surface immunoassay: a new approach to immunoanalysis with fluorescence detection.

This paper introduces a new methodology of carrying out heterogeneous immunoassays automatically, using a flow injection technique on a renewable surface. Flow injection renewable surface immunoassay (FIRSI) relies on the use of a minute amount of beads to form a reactive surface, which is interrogated by fluorescence spectrometry. Following the assay, on-line regeneration normally used in flow based immunoassays is avoided by fluidically removing the spent reactive surface and replacing it with a new layer of beads. This allows the monitoring of antibody-antigen binding at its early stages, dramatically increases the sampling frequency of a serial assay, and eliminates the problems associated with a decrease in surface reactivity caused by repetitive use. A model system utilizing anti-mouse IgG1-coated beads and mouse IgG1 protein is used to characterize the method with respect to reproducibility, flow rate, contact time, and amount of beads.

Animals↗
PubMed1993

Jet ring cell: a tool for flow injection spectroscopy and microscopy on a renewable solid support.

A new flow cell design for spectroscopic measurements of suspensions, the jet ring cell, is introduced. This cell exploits radial flow through a narrow ring-shaped gap to retain suspended particles within the detection region. This ring constitutes a detection volume of well-defined area from which the trapped particles can be instantaneously removed at will. The bed of particles thus forms a renewable surface, which can be probed by reflectance, fluorescence, or chemiluminescence using a microscope or optical fiber. This device should prove useful for microscopic study of cells, for automated immunoassays, and for preconcentration of analytes on sorbents with in situ spectroscopic detection. In conjunction with a fiber optic detection system, the jet ring cell becomes a component of a renewable chemical sensor system.

Animals↗
PubMed1993

Characterization of planar concentration gradients in a sequential-injection system for cell-perfusion studies.

This paper describes the characterization of a perfusion chamber that is coupled with a sequential-injection system and is being designed for live-cell perfusion. The apparatus consists of a multi-port valve, a peristaltic pump, a perfusion chamber and an epifluorescence microscope. The entire system is computer controlled and temperature regulated. The parameters discussed are the concentration-time profiles with regard to the volume of reagent used and the position of the cell in the perfusion chamber. Other parameters discussed include the stopped-flow compliance, reproducibility and symmetry of the concentration gradients formed. The system is shown to be suitable for two modes of perfusion; the first in which all cells are exposed to the same concentration of reagent, and the second in which cells are exposed to a gradient of concentrations. All characterization is performed with use of bulk fluorescein as a tracer, and a correlation is made between the bulk flow and the response within the cellular environment by using 5-[N-(octadecanoyl)amino]fluorescein.

Cells↗
PubMed1992

Sequential injection immunoassay utilizing immunomagnetic beads.

A novel sequential injection immunoassay (SIIA) method is described which utilizes immunomagnetic beads to investigate short-time antibody binding. The method is versatile and flexible and may therefore be adapted to many different applications. Initial results for a competitive assay are also presented. The immunomagnetic bead reactor is created within the flowing stream by retaining immunomagnetic beads with an electromagnet to form an open tube reactor. Thus, the spent beads may be discharged after each analysis. This eliminates the problems of instability of reaction surfaces and eliminates the need for additional time traditionally required for regeneration of the solid-reacting phase in order to not only save time and increase sampling frequency but also to provide each individual sampling cycle with a fresh, uniform portion of beads. The spent beads are collected off line and may be regenerated later. Short-time binding kinetic studies demonstrate linear initial binding under 1 min, which then begins to reach saturation in approximately 10 min. Competitive binding assays of monoclonal mouse IgG (MRC OX-19) to polyclonal sheep anti-mouse IgG immobilized to the immunomagnetic beads show reproducible linear displacement in 30-120-s reactions. Fluorescence detection is utilized with a detection limit of 155 ng/mL, and since the reaction time is typically 2 min or shorter, the sampling frequency is 30 samples/h.

Animals↗