Dissecting streptavidin-biotin interaction with a laminar flow chamber

A laminar flow chamber was used to study single molecule interactions between biotinylated surfaces and streptavidin-coated spheres subjected to a hydrodynamic drag lower than a piconewton. Spheres were tracked with 20 ms and 40 nm resolution. They displayed multiple arrests lasting between a few tens of milliseconds and several minutes or more. Analysis of about 500,000 positions revealed that streptavidin-biotin interaction was multiphasic: transient bound states displayed a rupture frequency of 5.3 s(-1) and a rate of transition toward a more stable configuration of 1.3 s(-1). These parameters did not display any significant change when the force exerted on bonds varied between 3.5 and 11 pN. However, the apparent rate of streptavidin-biotin association exhibited about 10-fold decrease when the wall shear rate was increased from 7 to 22 s(-1), which supports the existence of an energy barrier opposing the formation of the transient binding state. It is concluded that a laminar flow chamber can yield new and useful information on the formation of molecular bonds, and especially on the structure of the external part of the energy landscape of ligand-receptor complexes.     

Evaluation of laminar flow microbiological safety cabinets

The microbiological control efficiency of two class 100 laminar down-flow hoods was determined by using aerosols of Bacillus subtilis var. niger spores. The first unit challenged utilized a slanted eyelid to partially enclose the front work opening. This hood showed nearly perfect control of ambient organisms in the work area. It also gave a 10(6) or greater drop in the number of organisms passing out of the exhaust system. However, when the interior work area of the hood was challenged, significant numbers of spores penetrated the air barrier and escaped into the ambient air. A redesigned laminar flow hood was built incorporating a vertical eyelid and a reduced opening to the work area. This hood showed the same excellent characteristics for controlling ambient contamination. Exhaust system leakage was also extremely low. Air barrier efficiency for the newer hood was increased with lower amounts of spore penetration into the ambient air.     

Transglutaminase stabilizes melanoma adhesion under laminar flow

To resist substantial wall shear stress (WSS) exerted by flowing blood, metastatic melanoma cells can form adhesive contacts with subendothelial extracellular matrix proteins, such as fibronectin (FN). Such contacts may be stabilized by transglutaminase catalyzed-crosslinkage of cell focal adhesion proteins. We analyzed human melanoma cell adhesion under flow by decreasing the flow (WSS) of melanoma cell suspensions and allowing them to adhere to immobilized wheat germ agglutinin or FN. At the wall shear adhesion threshold (WSAT), cell adherence was rapid with no rolling. Following cell adherence, we increased the flow and determined the wall shear detachment threshold (WSDeT). Cells spread and remained adherent on immobilized FN at high WSDeTs (≥ 32.5 dynes/cm²). The high resistance of adherent cells to shear forces suggested that transglutaminase-mediated crosslinking might be involved. Transglutaminase inhibitors monodansylcadaverine and INO-3178 decreased WSAT, and at low concentrations completely inhibited tumor cell spreading and promoted detachment at low WSDeTs (0.67 dynes/cm²). In static adhesion assays, transglutaminase inhibitors decreased cell adhesion to immobilized-FN in a dose-dependent manner and prevented the formation of crosslinked¹²⁵I-FN complex that failed to enter a SDS-polyacrylamide gradient gel. The data suggest that transglutaminase-catalyzed crosslinking, particularly in the presence of WSS, may be important in stabilizing cellular adhesive contacts during adhesion to immobilized-FN.     

Performance of in-line microfluidic mixers in laminar flow for high-throughput flow cytometry

We describe a micromixing approach that is compatible with commercial autosamplers, flow cytometry, and other detection schemes that require the mixing of components that have been introduced into laminarflow. The scheme is based on high-throughput flow cytometry (HyperCyt) where samples from multi-well 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, either cell or particle samplesflowing continuously and driven by a syringe are brought together in a Y with reagent samples from wells driven by a peristaltic pump. The mixing is driven by a magnetic microstirrer contained within the sample line. The mixing is assessed using fluorescence of both cell calcium responses and bead-based fluorescence unquenching. In the analysis stream, the particles and reagents are mixed with eithera "wire" or "bar". The bar is more efficient than the wire, and the efficiency of either depends on the spinning action. The high-throughput approach and mixing in HyperCyt integrate autosamplers with submicroliter detection volumes for analysis in flow cytometry or in microfluidic channels.     

Transglutaminase stabilizes melanoma adhesion under laminar flow.

To resist substantial wall shear stress (WSS) exerted by flowing blood, metastatic melanoma cells can form adhesive contacts with subendothelial extracellular matrix proteins, such as fibronectin (FN). Such contacts may be stabilized by transglutaminase catalyzed-cross-linkage of cell focal adhesion proteins. We analyzed human melanoma cell adhesion under flow by decreasing the flow (WSS) of melanoma cell suspensions and allowing them to adhere to immobilized wheat germ agglutinin or FN. At the wall shear adhesion threshold (WSAT), cell adherence was rapid with no rolling. Following cell adherence, we increased the flow and determined the wall shear detachment threshold (WSDeT). Cells spread and remained adherent on immobilized FN at high WSDeTs (greater than or equal to 32.5 dynes/cm2). The high resistance of adherent cells to shear forces suggested that transglutaminase-mediated crosslinking might be involved. Transglutaminase inhibitors monodansylcadaverine and INO-3178 decreased WSAT, and at low concentrations completely inhibited tumor cell spreading and promoted detachment at low WSDeTs (0.67 dynes/cm2). In static adhesion assays, transglutaminase inhibitors decreased cell adhesion to immobilized-FN in a dose-dependent manner and prevented the formation of crosslinked 125I-FN complex that failed to enter a SDS-polyacrylamide gradient gel. The data suggest that transglutaminase-catalyzed crosslinking, particularly in the presence of WSS, may be important in stabilizing cellular adhesive contacts during adhesion to immobilized-FN.     

Heat transport in laminar flow of erythrocyte suspensions.

Measurements of thermal conductivity were made in laminar flow of dog and turkey erythrocyte suspensions in a stainless stell tube of about 1 mm ID. These measurements were independent of the shear rate, showing that the red cell motion relative to plasma in flowing blood had no effect on the heat transfer. Measurements of thermal conductivity were further made in suspensions of polystyrene spheres of 100 mum and were found to be dependent upon the shear rate. The Graetz solution corresponding to uniform wall temperature was used for determining the value of thermal conductivity in an apparatus calibrated with tap water. The overall accuracy of the results is within 10%. A model based on the particle rotation with the entrained fluid is proposed. It is pointed out that the diffusion of platelets, red cells, and possibly plasma proteins (such as fibrinogen) will be augmented if they happen to be in the hydrodynamic field of rotating erythrocytes.     

Axial migration of spirulina microalgae in laminar tube flow

A dilute suspension of Spirulina Microalgae is found to exhibit radial migration in laminar flow in a 650 micron vertical tube. As the tube Reynolds number increases, the particles concentrate in a narrower region around the tube axis. When the turbulent regime is approached, the particles disperse as expected.     

Mathematical model of the distribution of small negative ions in air conditioned rooms

The distribution of small negative ions with mobility k_r=0.5+ (cm²/Vs) in an air conditioned room is presented. The air was ionized by an ion generator, which was placed at the outlet of the air duct. The measurements were made at 100 different locations in the room. It was established, that the main factors in the distribution of ions were the coordinates of points in the room space. Regression analysis method was applied to determine optimal estimate of identified static characteristics of ions distribution. The main purpose of this research was to obtain data for the correct placement of ion generators in air conditioned or ventilated rooms.     

Developing steady laminar flow through uniform straight tubes with varying wall cross curvature

Numerical calculations are used to determine not only the wall shear stress but also the entry length in a laminar steady flow of an incompressible Newtonian fluid. The fluid is conveyed through rigid straight tubes with axially uniform cross sections, which mimic collapsed vessels. For each tube configuration, the "Navier-Stokes" equations are solved using the finite element method. The numerical tests are performed with the same value of the volume flow-rate whatever the tube configuration for three "Reynolds numbers". The wall shear stress is computed and determined along the axis of the tube, then the entry length is estimated by introducing two indexes by using: (i) the axial fluid velocity, and (ii) the wall shear stress. The results are analysed in order to exhibit the mechanical environment of cultured endothelial cells in the flow chamber for which the test conditions will be well-defined. For example, in a tube configuration where the opposite walls are in contact for which the inner perimeter and the area of the cross section are respectively given by 45 mm and 37.02 mm(2), the computed entry lengths with the criteria defined by (i) and (ii) are equals to about 118 and 126 mm, respectively for R(e0) = 500.     

Normal values of peak expiratory flow in children from an environment with high degree of atmospheric air pollution]

Peak expiratory flow (PEF) has been measured with Vitalograph (in liters per minute) in 2,512 school-children aged between 7 and 15 years in Upper Silesian Industrial Region. Five hundred eighty one children from Zarki near Czestochowa served as a control group. The results have been analysed statistically. Determined PEF values for children from the Upper Silesian Industrial Region are considered as a biological reference values for assessment of PEF in both health and disease. PEF values calculated for children from Zarki were higher than those in the examined group, except the value for a 14-year old children.     

Biofilm formation in laminar flow usingPseudomonas fluorescens EX101

The relationship between biofilm formation and Reynolds number in laminar flow has been investigated usingPseudomonas fluorescens EX101. It was shown using a Modified Robbins Device that in laminar flow, numbers of viable cells in a developed biofilm increased with Reynolds number (Re 2, 17 and 51.5), as would be expected in a system where molecular transport to the wall is limited by diffusion. By monitoring fluorescent beads in a flowcell with a scanning confocal laser microscope at similar low Reynolds numbers, the velocity profile close to the solid surface was determined. It was shown that the presence of a thin bacterial film (up to 12 m) displaced the flow profile away from the wall by a distance equivalent to the film thickness. Total cell counts from the Modified Robbins Device samples were not significantly different at the different flow rates but were higher than viable counts. Interruption of the flow had no significant effect on colonisation by the bacteria through the Modified Robbins Device in the first few hours. However, viable numbers were reduced when the flow was stopped at 7 h after initial colonisation.     

The use of laminar flow for obtaining germ-free mice.

Hysterectomy in a laminar-flow cabinet affords greater economy of time and effort in obtaining germ-free mice than other methods. No contamination has occurred during the surgical procedure or in the subsequent transfer of the neonates into germ-free isolators.     

Bacteriological testing of a modified laminar flow microbiological safety cabinet

A modified microbiological safety cabinet which can be used as a class II and a class III safety cabinet has been bacteriologically tested. This cabinet makes use of a high-speed down-flow air curtain in the front opening to minimize the amount of air escaping over the arms of the operator. By using artificial aerosols and a dummy or a test person placing his arms into the working opening of the cabinet, a transfer from the inside to the environment was detected only when the highest concentration of the test aerosol was used. Since the number of bacteria detected was very low, this is considered to be acceptable. when the cabinet was used as a class III type, with a glove panel mounted in the front opening, leakage from the environment occurred. This could be completely prevented by fixing tape over the hinge of the front panel.The conclusion is drawn that this type of biohazard hood can be safely used as a class II and a class III microbiological safety cabinet, provided the construction of the hinge of the front panel will be adapted to prevent transfer from the environment to the working area.     

Fractionation of cell mixtures using acoustic and laminar flow fields

A fractionation method applicable to different populations of cells in a suspension is reported. The separation was accomplished by subjecting the suspension to a resonant ultrasonic field and a laminar flow field propagating in orthogonal directions within a thin, rectangular chamber. Steady, laminar flow transports the cell suspension along the chamber, while the ultrasonic field causes the suspended cells to migrate to the mid-plane of the chamber at rates related to their size and physical properties. A thin flow splitter positioned near the outlet divides the effluent cell suspension into two product streams, thereby allowing cells that respond faster to the acoustic field to be separated from those cells that respond more slowly. Modeling of the trajectories of individual cells through the chamber shows that by altering the strength of the flow relative to that of the acoustic field, the desired fractionation can be controlled. Proof-of-concept experiments were performed using hybridoma cells and Lactobacillus rhamnosus cells. The two populations of cells could be effectively separated using this technique, resulting in hybridoma/Lactobacillus ratios in the left and right product streams, normalized to the feed ratio, of 6.9 +/- 1.8 and 0.39 +/- 0.01 (vol/vol), respectively. The acoustic method is fast, efficient, and could be operated continuously with a high degree of selectivity and yield and with low power consumption. (c) 2004 Wiley Periodicals, Inc.