Flow cell assay

In the Biacore AlOO system containing four FIA flow cells with five immo-bihzation spots per flow cell, assays can be designed either for the maximum number of samples, or to deliver the maximiun information per sample. In the first instance, identical immobihzations may be performed in all four flow cells, allowing four different samples to be analyzed in parallel during each analysis cycle, while in the latter, up to 20 different interactants may be immo-bihzed across all four flow cells with one sample per cycle injected in parallel. 

It is important to note that antimicrobial and biofilm resistance are two different characteristics though some materials show both properties at the same time. Antimicrobial materials do not automatically prevent biofilm formation and vice versa. Antimicrobial surfaces could kill bacteria on contact but if dead bacteria cell debris blocks the active biocidal surface, biofilm formation could eventually occur. For example, quaternary anunonium polymers can effectively kill bacteria but when the surface is fouled with dead bacteria debris, biofilm formation is inevitable [188]. Materials with antibiofilm properties will repel the bacterial adhesion very effectively but may not kill the bacteria when they do colonize the surface. PEG surfaces are well known to repel bacteria adhesion. However, PEG surfaces show little antimicrobial activity. Quantitative antibiofilm efficacy tests can be divided into two categories static (minimum biofilm eradication concentration assay, MBEC) and dynamic (flow cell assay). In addition, SEM is a semiquantitative assay, which is discussed in Section 2.5. 

A typical flow cell assay involves a flow cell chamber, a microscope (white light or fluorescent), a peristaltic pump, and connection tubings. The basic construct of a flow cell is shown in Figure 2.38. The flow chamber is sandwiched between two optical clear plates (glass or plastic). Based on test specification, the flow chamber... 

Luminescence has been used in conjunction with flow cells to detect electro-generated intennediates downstream of the electrode. The teclmique lends itself especially to the investigation of photoelectrochemical processes, since it can yield mfonnation about excited states of reactive species and their lifetimes. It has become an attractive detection method for various organic and inorganic compounds, and highly sensitive assays for several clinically important analytes such as oxalate, NADH, amino acids and various aliphatic and cyclic amines have been developed. It has also found use in microelectrode fundamental studies in low-dielectric-constant organic solvents. 

Compounds that are not overtly myelotoxic may still selectively damage or destroy lymphocytes, which are the primary effectors and regulators of acquired immunity. This toxicity may result from the destruction of rapidly dividing cells by necrosis or apoptosis. A variety of methodologies are available for this purpose (e.g., colorimetric, flow cytometric assays). If the cells are viable (80% or greater), basic functionality could be determined by performing specific functional assays. 

Manetz, T.S. and Meade, B J., Development of a flow cytometry assay for the identification and differentiation of chemicals with the potential to elicit irritation, IgE-mediated, or T cell-mediated hypersensitivity responses, Toxicol. Sci.. 48, 206, 1999. 

Some, uPLC systems are equipped with UV absorbance detection, and other systems allow for both UV absorbance and fluorescence detection. Fluorescence detection increases the sensitivity and selectivity of certain applications and is the method of choice in many separation-based assays. The liquid (mobile phase + sample) leaving the individual flow cells designated for UV detection is transferred through capillaries to a bank of 24 flow cells designated for fluorescence detection. 

Two-dimensional (2D) separation systems in microfluidic assays, 26 970 Two-dimensional (2D) transistor strustures, in scaling to deep submicron dimensions, 22 256 Two-dimensional data searches, 6 6-8 Two-dimensional electrode flow cells, 9 664-665... 

Unfortunately monoparametric DNA content analysis by PI is not able to discriminate from different cell cycle phases, and as exemplified in Figure 4, monoparametric analysis by PI show its limitations, since this flow cytometric assay does not display any details S-phase activities after drug treatments. At this point, cells can be arrested in a specific cell phase (eg. Gi or G2/M) and obviously a decrease of S-phase is observed. 

Black CB, Duensing TD, Trinkle LS, Dunlay RT. Cell-based screening using high-throughput flow cytometry. Assay Drug Dev. Technol. 2010 Feb 9(l) 13-20. 

Mehes G, Pajor L (1995) Nucleolin and fibrillarin expression in stimulated lymphocytes and differentiating HL-60 cells. A flow cytometric assay. Cell Prolif 28 329-336 Nasirudin KM, Ehtesham NZ, Tuteja R, Sopory SK, Tuteja N (2005) The Gly-Arg-rich C-terminal domain of pea nucleolin is a DNA helicase that catalytically translocates in the 5 - to 3 -direction. Arch Biochem Biophys 434 306-315... 

In some instances, flow cytometry assays are a superior alternative to conventional procedures for the determination of equilibrium binding constants (Stein et al., 2001). In contrast to assays that employ radiolabelled ligands, which measure population mean values for binding constants, flow cytometry methods can measure those values in individual cells, revealing heterogeneity in receptor expression within a population of cells or membrane vesicles. Furthermore, small samples can be characterized in a short period of time (hours). This approach to receptor-binding analysis may be limited only by the availability of a properly characterized fluorescent ligand. 

A variety of assays have been developed to quantify phagocytic activity. These include direct microscopic visualization (2,3), spectrophotometric evaluation of phagocytized paraffin droplets containing dye (4), scintillation counting of radiolabeled bacteria (5), fluorometric (6), and flow cytometric analysis of fluorescent particles (7-13). The flow cytometric assay offers the advantage of rapid analysis of thousands of cells and quantification of the internalized particle density for each analyzed cell. The assay may be performed with purified leukocyte preparations (7-13) or anficoagulated whole blood (14,15). 

Molecules (chemoattractants) that stimulate neutrophil-directed migration (chemotaxis) bind to distinct receptors on neutrophil plasma membranes (discussed in Chapter 38 of this text). Within seconds after chemoattractant binding, neutrophils exhibit rapid oscillations in actin polymerization and depolymerization (12,13). The shape changes accompanying chemoattractant binding depend on the duration and extent of F-actin polymerization (3). These quantitative studies of F-actin content were performed utilizing a flow cytometric assay that detects the fluorescence intensity of individual, fixed, permeabihzed cells that have been stained with F-actin-specific, fluorescent phallotoxins (14,15). 

Biochemical oxygen demand (BOD) is one of the most widely determined parameters in managing organic pollution. The conventional BOD test includes a 5-day incubation period, so a more expeditious and reproducible method for assessment of this parameter is required. Trichosporon cutaneum, a microorganism formerly used in waste water treatment, has also been employed to construct a BOD biosensor. The dynamic system where the sensor was implemented consisted of a 0.1 M phosphate buffer at pH 7 saturated with dissolved oxygen which was transferred to a flow-cell at a rate of 1 mL/min. When the current reached a steady-state value, a sample was injected into the flow-cell at 0.2 mL/min. The steady-state current was found to be dependent on the BOD of the sample solution. After the sample was flushed from the flow-cell, the current of the microbial sensor gradually returned to its initial level. The response time of microbial sensors depends on the nature of the sample solution concerned. A linear relationship was foimd between the current difference (i.e. that between the initial and final steady-state currents) and the 5-day BOD assay of the standard solution up to 60 mg/L. The minimum measurable BOD was 3 mg/L. The current was reproducible within 6% of the relative error when a BOD of 40 mg/L was used over 10 experiments [128]. 

---