A sample injection device for flow cytometers

BACKGROUND: The sample injection systems of flow cytometers employ either a pressure differential between the sample vial and the sheath fluid reservoir or volumetric injection of the sample from a syringe. The pressure differential method facilitates rapid and efficient flushing to eliminate carryover between samples, but does not allow accurate determination of the rate of sample flow and cell concentration. Volumetric injection, which comprises a valve for switching the sample flow, facilitates highly accurate measurement of the cell concentration, but requires a less efficient and more time-consuming flushing procedure. METHODS: Applying a removable syringe, which connects to the inlet of the sample tubing via a tight sealing, we eliminate the valve and obtain efficient flushing while maintaining the advantage of volumetric sample injection. RESULTS: This device gives highly constant sample flow rates strictly proportional to syringe velocity over the range 0.2-50 microl/min with a settling time of about 2 sec. CONCLUSION: This device has the same precision as the conventional sample injection system, whereas the speed and efficiency of flushing are improved greatly.     

Dye exclusion artifact in flow cytometers

BACKGROUND: Cells exclude their own volume of dye solution in the sample flow which carries them through the flow chamber of the flow cytometer, thereby affecting the otherwise constant signal arising from the fluorescence of this solution. Under certain conditions, this phenomenon may significantly influence the fluorescence signal of the cells. MATERIALS AND METHODS: Using the slit scan technique, we studied this phenomenon as observed for monodisperse polystyrene particles in fluorescein solution. RESULTS: The measurements show that dye solution accumulates just in front of the particle and just behind it, with a relative void in between. This phenomenon is most likely caused by the rapid constriction of the flow as it enters the orifice of the nozzle or flow chamber, giving rise to a pulse of fluorescence which adds to that of the particle or cell itself. The magnitude of this artifact depends on the design and dimensions of the nozzle/flow chamber as well as on the rate of sample flow. CONCLUSIONS: The dye exclusion artifact may affect measurements of cells when they are in a dye solution having a fluorescence per unit volume which is significant compared to that of the cells, especially at low sample flow rates.     

A collection device for small electronically sorted samples from flow cytometers

A device (Hunziker chamber) has been fabricated which facilitates the collection and biochemical analysis of electrostatically sorted nuclei. The G₀ + G₁ and G₂ + M populations and the 2N and 4N populations of rat liver nuclei were sorted on the basis of light scatter or fluorescence by the fluorescent activated cell sorter (Becton-Dickinson). Sufficient amounts of nuclear samples were sorted and collected in the Hunziker chamber for the analysis of nuclear proteins by sodium dodecyl sulfate discontinuous polyacrylamide gel electrophoresis. An example of the electrophoretic banding patterns of nuclear proteins from these populations is given.     

A comparison of mercury arc lamp and laser illumination for flow cytometers.

Optical differences between a mercury arc lamp and a laser-illuminated flow cytometer are compared. The distributions of spectral intensities of the two light sources are shown in relation to the excitation characteristics of the fluorescent dyes acriflavine, chromomycin A3, mithramycin, ethidium bromide, Hoechst 33258, and 4,6-diamidino-2-phenylindole (DAPI). Fluorescence intensities of microspheres and Hoechst 33258-stained mouse sperm are compared in the two cytometers. The optical efficiencies are similar and depend on the match of the excitation characteristics of the stain with the emission spectra of the light source.     

Antibody-functionalized fluid-permeable surfaces for rolling cell capture at high flow rates

Adhesion-based cell capture on surfaces in microfluidic devices forms the basis of numerous biomedical diagnostics and in vitro assays. However, the performance of these platforms is partly limited by interfacial phenomena that occur at low Reynolds numbers. In contrast, cell homing to porous vasculature is highly effective in vivo during inflammation, stem cell trafficking, and cancer metastasis. Here, we show that a porous, fluid-permeable surface functionalized with cell-specific antibodies promotes efficient and selective cell capture in vitro. This architecture is advantageous due to enhanced transport as streamlines are diverted toward the surface. Moreover, specific cell-surface interactions are promoted due to reduced shear, allowing gentle cell rolling and arrest. Together, these synergistic effects enable highly effective cell capture at flow rates more than an order of magnitude larger than those provided by existing devices with solid surfaces.     

A comparison of low-power helium-cadmium and argon ultraviolet lasers in commercial flow cytometers

The feasibility of installing a low power ultraviolet (UV) laser in a commercial flow cytometer was evaluated by testing an Ortho Cytofluorograf 50HH and a Coulter Epics V. Both instruments were equipped with two argon ion lasers, one emitting at 488 nm and the other in the UV region and were tested by measuring the DNA content of cells stained with Hoechst 33342 or DAPI. The coefficient of variation (CV) of the G0/G1 peak of the DNA histograms produced by each instrument did not deteriorate markedly when results obtained at 100-125 mW were compared to those obtained at 10 mW. These tests indicated that a helium-cadmium laser (He-Cd) which can produce 10 mW at 325 nm should work well as a UV laser in these instruments. An Ortho Cytofluorograf IIs was purchased with a 10 mW He-Cd laser installed in the forward position. Studies of DNA content have confirmed that this low power UV laser can produce CVs of 2.2% with DAPI stained fixed cells and 3.6% with Hoechst 33342 stained viable lymphocytes. Thus, the He-Cd laser should provide a reasonable alternative as a UV source for flow cytometers.     

Sample station modification providing on-line reagent addition and reduced sample transit time for flow cytometers

A flow cytometer was equipped with a modified sample station to facilitate on-line addition of mediators to the sample and reduce the time of delivery of the sample to the interrogation point. The ready availability of materials and straightforward nature of this design make modifications simple and facilitate measurements of cellular activation. Parameters such as pH, membrane fluidity, and calcium mobilization are easily measured in this system, because detection can be made less than 4 s after addition of mediator with no interruption of sample flow. The sample station modification is described in detail along with methods for mixing and temperature control.     

A low shear stress modular bioreactor for connected cell culture under high flow rates

A generic “system on a plate” modular multicompartmental bioreactor array which enables microwell protocols to be transferred directly to the bioreactor modules, without redesign of cell culture experiments or protocols is described. The modular bioreactors are simple to assemble and use and can be easily compared with standard controls since cell numbers and medium volumes are quite similar. Starting from fluid dynamic and mass transport considerations, a modular bioreactor chamber was first modeled and then fabricated using “milli‐molding,” a technique adapted from soft lithography. After confirming that the shear stress was extremely low in the system in the range of useful flow rates, the bioreactor chambers were tested using hepatocytes. The results show that the bioreactor chambers can increase or maintain cell viability and function when the flow rates are below 500 µL/min, corresponding to wall shear stresses of 10^?5 Pa or less at the cell culture surface. Biotechnol. Bioeng. 2010; 106: 127–137. © 2010 Wiley Periodicals, Inc.     

Time-intensity evaluation of acid taste in subjects with saliva high flow and low flow rates for acids of various chemical properties

The role of the chemical properties of sour stimuli and the role of the human saliva flow rate on acid perception were investigated in 11 high saliva flow rate (HF) and 11 low saliva flow rate (LF) subjects with a continuous stimulus delivery flow rate of 3.2 ml/min and using the time-intensity technique for perception recording. Continuously measuring the pH on the tongue surface on three HF and three LF subjects showed that HF subjects' saliva decreased the acidity of the acid solution more efficiently than the LF subjects' saliva did. However, HF subjects exhibited higher perceived intensity for acid solutions than LF subjects. At equal pH, the order of the efficiency of acids indicated that HCl was the least efficient acid stimulus and acetic acid the most efficient. At equal concentration, the order of efficiency was the opposite (citric acid > malic acid > lactic acid > acetic acid), indicating that titratable acidity rather than pH has to be considered when comparing weak acids. At high concentrations, the ratio of relative efficiency is more in favor of the hydrophobic than the hydrophilic acid in HF subjects compared with LF subjects, i.e. HF subjects are more sensitive to hydrophobic stimuli. Hydrophobic molecules may diffuse more easily into the epithelium of HF than LF subjects, and reach more efficiently trigeminal nerve endings in addition to taste receptor cells.     

Sinks for photosynthetic electron flow in green petioles and pedicels of Zantedeschia aethiopica: evidence for innately high photorespiration and cyclic electron flow rates

A combination of gas exchange and various chlorophyll fluorescence measurements under varying O₂ and CO₂ partial pressures were used to characterize photosynthesis in green, stomata-bearing petioles of Zantedeschia aethiopica (calla lily) while corresponding leaves served as controls. Compared to leaves, petioles displayed considerably lower CO₂ assimilation rates, limited by both stomatal and mesophyll components. Further analysis of mesophyll limitations indicated lower carboxylating efficiencies and insufficient RuBP regeneration but almost similar rates of linear electron transport. Accordingly, higher oxygenation/carboxylation ratios were assumed for petioles and confirmed by experiments under non-photorespiratory conditions. Higher photorespiration rates in petioles were accompanied by higher cyclic electron flow around PSI, the latter being possibly linked to limitations in electron transport from intermediate electron carriers to end acceptors and low contents of PSI. Based on chlorophyll fluorescence methods, similar conclusions can be drawn for green pedicels, although gas exchange in these organs could not be applied due to their bulky size. Since our test plants were not subjected to stress we argue that higher photorespiration and cyclic electron flow rates are innate attributes of photosynthesis in stalks of calla lily. Active nitrogen metabolism may be inferred, while increased cyclic electron flow may provide the additional ATP required for the enhanced photorespiratory activity in petiole and pedicel chloroplasts and/or the decarboxylation of malate ascending from roots.     

Chromatography of hen egg-white proteins on anion-exchange cellulose at high flow rates using a radial flow column.

The influence of column configuration on the separation of hen egg-white proteins using Whatman DE52 and QA52 anion-exchange cellulose has been investigated. Using a 100 ml volume axial flow column (6.6 cm x 4.4 cm i.d.) we achieved flow rates of up to 25 ml/min i.e. 15 bed volumes/h after which higher flow was restricted due to pressure constraints within the system. Under radial flow conditions using a 100 ml column flow rates of up to 150 ml/min i.e. 90 bed volumes/h were achieved using DE52 and QA52. While chromatographic resolution was superior under axial flow at the lower flow rates excellent resolution was maintained at up to 150 ml/min using the radial flow column. This is a consequence of the fast kinetics of adsorption/desorption exhibited by DE52 and QA52. The data indicate that it is the column configuration and not the cellulose matrix which influences flow performance.     

A novel cyclic opioid peptide analog showing high preference for mu-receptors

The side-chain to side-chain cyclized opioid peptide analogs H-Tyr-D-Orn-Phe-Asp-NH2 (I) and H-Tyr-D-Lys-Phe-Glu-NH2 (II) were synthesized and tested in the guinea pig ileum and mouse vas deferens assays and in binding assays based on displacement of mu- and delta-opioid receptor-selective radioligands from rat brain membranes. The more rigid cyclic analog I containing a 13-membered ring structure showed very high preference for mu-receptors over delta-receptors, whereas the more flexible cyclic peptide II (15-membered ring) was non-selective. These results indicate that variation in the degree of conformational restriction of opioid peptides can produce drastic shifts in their receptor selectivity profile. Because of its high mu-receptor selectivity and rigidity cyclic analog I will be useful for determining the conformational requirements of mu-opioid receptors.     

Determination of division rates of rCHO cells in high density and immobilized fermentation systems by flow cytometry

As most high density and immobilized fermentation systems do not allow the direct quantitative determination of cell density, two flow cytometric methods (the determination of incorporation of bromodeoxyuridine into newly synthesized DNA and the increase in mitotic cells by colchicine blockage) were evaluated as to their suitability to measure true division rates of cells in bioreactors. The BrdU method gave division rates identical to the growth rates measured by cell count, while the colchicine block method gave values that were lower and varied with the cell line. This is due to the cytotoxicity of colchicine and makes a calibration of the method for each cell line necessary. Both methods have been successfully used to measure division rates of rCHO cells immobilized in an alginate matrix as well as in macroporous carriers in a fluidised bed system and in dialysis culture.     

Flow cytometers using optical waveguides in place of lenses for specimen illumination and light collection

Optical waveguides represent alternatives to lenses for delivery of light to, and collection of light from, small regions of space such as the observation point(s) of a flow cytometer. The flow chamber and directly associated illumination and collection optics of a flow cytometer can be built using fiber optic waveguides in fixed positions or as an integrated optical assembly incorporating deposited or implanted waveguides, providing illumination and collection geometries and efficiencies comparable to conventional designs, with the advantage that the optical waveguide design, once assembled, never again requires alignment of the optics. A prototype apparatus in which fiber optics were used to illuminate and collect light from a point inside a thin-walled, round quartz capillary was observed to measure fluorescence of polystyrene spheres with precision close to that obtained from a conventional flow cytometer using the same laser source. The design of the fiber optic system is readily adaptable to construction of multiple illumination beam instruments; other optical waveguide flow cytometer designs that dispense with the capillary share this advantage and can also provide orifices for electronic volume sensing and for jetting prior to droplet sorting.     

Anisotropy of stationary rates and delays in extrasystolic waves in the dog heart]

Excitation propagation in the ventricle and auricle bands of the dog's myocardium has been studied. The anisotropy of stationary valocities and delays of the initiation of extrasistole exciation has been shown: along the fibres the velocity is higher and the delay smaller than across the fibres. The levels of anisotropy of velocities and delays are almost equal in each structure. The strongest anisotropy of delays and velocities (-4,5) is observed in the ventricle muscle layer; the weakest (1,7)-in a specialized layer. The anisotropy of velocities and delays is the greater, the greater is the electrotone anisotropy. A new mechanism of extrasistole initiation (reentry) in the system homogenous by the membrane properties, but with anisotropy of intercellular bonds is considered.     

Active involvement of PKC for insulin-mediated rates of muscle protein synthesis in Zucker rats

A recent report from our group demonstrated that insulin facilitates muscle protein synthesis in obese Zucker rats. The purpose of this study was to determine whether PKC, a probable modulator of insulin signal transduction and/or mRNA translation, has a role in this insulin-mediated anabolic response. In the first portion of the study, gastrocnemius muscles of lean and obese Zucker rats (n = 5-7 for each phenotype) were bilaterally perfused with or without insulin to assess cytosolic and membrane PKC activity. Limbs perfused with insulin demonstrated greater PKC activity in both lean and obese Zucker rats (P < 0.05) compared with no insulin, but overall activity was greater in obese animals (by approximately 27% compared with lean, P < 0.05). To determine whether PKC plays a role in muscle protein synthesis, hindlimbs (n = 6-8 for each phenotype) were bilaterally perfused with or without insulin and/or GF-109203X (GF; a PKC inhibitor). The presence of GF did not influence the rates of insulin-mediated protein synthesis in gastrocnemius muscle of lean Zucker rats. However, when obese rats were perfused with GF (P < 0.05), the effect of insulin on elevating rates of protein synthesis was not observed. We also used phorbol 12-myristate 13-acetate (TPA, a PKC activator; n = 5-7 for each phenotype) with and without insulin to determine the effect of PKC activation on muscle protein synthesis. TPA alone did not elevate muscle protein synthesis in lean or obese rats. However, TPA plus insulin resulted in elevated rates of protein synthesis in both phenotypes that were similar to rates of insulin alone of obese rats. These results suggest that PKC is a modulator and is necessary, but not sufficient, for insulin-mediated protein anabolic responses in skeletal muscle.     

Rapid parasite adaptation drives selection for high recombination rates

The Red Queen hypothesis proposes that sex is maintained through selection pressure imposed by coevolving parasites: susceptible hosts are able to escape parasite pressure by recombining their genome to create resistant offspring. However, previous theoretical studies have shown that the Red Queen typically selects against sex unless selection is strong, arguing that high rates of recombination cannot evolve when parasites are of low virulence. Here we show that under the biologically plausible assumption of a severe fitness cost for parasites that fail to infect, the Red Queen can cause selection for high recombination rates, and that the strength of virulence is largely irrelevant to the direction of selection for increased recombination rates. Strong selection on parasites and short generation times make parasites usually better adapted to their hosts than vice versa and can thus favor higher recombination rates in hosts. By demonstrating the importance of host-imposed selection on parasites, our findings resolve previously reported conflicting results.