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.     

Safe sorting of GFP-transduced live cells for subsequent culture using a modified FACS vantage.

BACKGROUND: A stream-in-air cell sorter enables rapid sorting to a high purity, but it is not well suited for sorting of infectious material due to the risk of airborne spread to the surroundings. METHODS: A FACS Vantage cell sorter was modified for safe use with potentially HIV infected cells. Safety tests with bacteriophages were performed to evaluate the potential spread of biologically active material during cell sorting. Cells transduced with a retroviral vector carrying the gene for GFP were sorted on the basis of their GFP fluorescence, and GFP expression was followed during subsequent culture. RESULTS: The bacteriophage sorting showed that the biologically active material was confined to the sorting chamber. A failure mode simulating a nozzle blockage resulted in detectable droplets inside the sorting chamber, but no droplets could be detected when an additional air suction from the sorting chamber had been put on. The GFP transduced cells were sorted to 99% purity. Cells not expressing GFP at the time of sorting did not turn on the gene during subsequent culture. Un-sorted cells and cells sorted to be positive for GFP showed a decrease in the fraction of GFP positive cells during culture. CONCLUSIONS: Sorting of live infected cells can be performed safely and with no deleterious effects on vector expression using the modified FACS Vantage instrument.     

Separation of large quantities of chinese hamster chromosomes by velocity sedimentation at unit gravity followed by flow sorting (FACS II)

Separation of large quantities of isolated metaphase chromosomes of Chinese hamster cells was performed by velocity sedimentation at unit gravity in a specially designed sedimentation chamber. This simple and easy technique results in chromosome fractions of relatively high purity as determined by flow cytometry and microscopy. Up to 10¹⁰ chromosomes can be processed depending upon the size of the sedimentation device, and enrichments up to 10 times of individual chromosomes were achieved. In addition, further chromosome purification was performed by fluorescence activated flow sorting using fractions, pre-enriched at unit gravity. The flow sorted chromosomal fractions were pure according to flow cytometric analyses. The combination of l g sedimentation and flow-sorting opens the possibility for preparative chromosome sorting by reducing the flow sorting time considerably.     

Flow cytometry chamber with 4 pi light collection suitable for epifluorescence microscopes

A compact, solid, spherico-ellipsoidal chamber (SEC), which has approaching 4 pi ("all around") light collection, has been developed for flow cytometry. This was mounted onto the stage of a standard fluorescence photomicroscope, and the camera was replaced by a photomultiplier. Both components can be added or removed in minutes. The increased light collection efficiency of the SEC (about 85%) compared with about 4% from standard chambers enabled a fluorescence microscope with a 50 W mercury vapour lamp to "double" as a flow cytometer. The system was tested with microbeads and cells stained for DNA with ethidium bromide, and results were comparable to those obtained with our laser-based instrument.     

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.     

Modification of a laser-based flow cytometer for high-resolution DNA analysis of mammalian spermatozoa

Modification of a Coulter EPICS V orthogonal laser-based flow cytometer/cell sorter allows resolution of X and Y mammalian sperm populations based on DNA content. The modification consists of beveling the sample injection tube situated in the flow chamber, adding a second fluorescence detector directly forward along the laser beam axis, and routing the collected fluorescence through an optical fiber bundle to one of the existing photomultiplier tubes. The X and Y chromosome-bearing spermatozoa from the bull, boar, and ram can be resolved using this system.     

High-throughput droplet PCR

The polymerase chain reaction has facilitated the ready analysis of nucleic acids. A next challenge requires the development of means to unravel the complexity of heterogeneous tissues. This has presented the task of producing massively parallelized quantitative nucleic acid data from the cellular constituents of tissues. The production of aqueous droplets in a two phase flow is shown to be readily and routinely facilitated by miniaturized fluidic devices. Droplets serve as ideal means to package a future generation of PCR, offering an enhanced handling potential by virtue of reactant containment, to concurrently eliminate both contamination and sample loss. This containment also enables the measurement of nucleic acids from populations of cells, or molecules by means of high throughput, single cell analysis. Details are provided for the production of a prototype micro-fluidic device which shows the production and stable flow of droplets which we suggest will be suitable for droplet-based continuous flow micro-fluidic PCR. Suggestions are also made as to the optimal fabrication techniques and the importance of device calibration.     

A microscope-based flow cytophotometer

By means of a new flow chamber, a standard fluorescence microscope with Epi illumination and 100 W mercury arc excitation has been turned into a flow cytophotometer combining high resolution and sensitivity with simplicity of operation. In the flow chamber, cells are passed in a narrow stream through the microscope focus carried by a laminar flow of water running on the open surface of a cover glass which is coupled to the oil immersion microscope objective. Two spectral components of the fluorescence, for example, resulting from specific staining of two different cellular constituents with different dyes, can be measured simultaneously in separate channels so as to produce three-dimensional histograms. The scattered light of the cells is detected in dark field by a second microscope situated opposite the primary objective. Scattered light detection is integrating with regard to scattering angle from 0 degree to 90 degrees. Hence, diffraction pattern effects are eliminated and the light scatter signal is approximately proportional to cell dry weight. The Epi illumination, which implies that excitation and fluorescence collection are parfocal, greatly simplifies instrument adjustment, which is further facilitated by the fact that the cell stream can be viewed at high magnification. Cell measuring time is about 3 microseconds which implies a measuring rate of 3 x 10(3) cells/s at 1% coincidence rate. Sensitivity is sufficient for measuring the DNA content of bacteria (that is, approximately 5 x 10(-15) g/cell) with a coefficient of variance (CV) of about 6%. CV less than 1% is achieved for DNA histograms of mammalian cells. A 5 W argon laser as excitation source facilitates slit scan analysis and increases the sensitivity and measuring rate by one to two orders of magnitude.     

Multiparameter analysis and sorting of mammalian cells

An improved multisensor cell sorting instrument for quantitative analysis and sorting of cells has been developed. Cells stained with fluorescent dyes enter a flow chamber where cell volume, fluorescence, and light scatter sensors simultaneously measure multiple cellular properties. Cells then emerge in a liquid jet that is broken into uniform liquid droplets. Sensor signals are electronically processed in one of several ways for optimum cell discrimination and are displayed as pulse-amplitude distributions using a multichannel pulse-height analyzer. Processed signals activate cell sorting according to preselected parametric criteria by electrically charging droplets containing cells and electrostatically deflecting them into collection vessels. Illustrative examples of multiparameter cell analysis and sorting experiments using a model mouse tumor cell system, human and animal leukocytes, and cultured mammalian cells are presented.     

Flow microfluorometric and light-scatter measurement of nuclear and cytoplasmic size in mammalian cells.

A technique for rapid measurement of nuclear and cytoplasmic size relationships in mammalian cell populations has been developed. Based on fluorescence staining of either the nucleus alone or in combination with the cytoplasm using two-color fluorescence methods, this technique permits the simultaneous determination of nuclear and cytoplasmic diameters from fluorescence and light-scatter measurements. Cells stained in liquid suspension pass through a flow chamber at a constant velocity, intersecting a laser beam which excites cell fluorescence and causes light scatter. Depending upon which analysis procedure is used, optical sensors measure nuclear fluorescence and light scatter (whole cell size) or two-color nuclear and cytoplasmic fluorescence from individual cells crossing the laser beam. The time durations of signals generated by the nucleus and cytoplasm are converted electronically into signals proportional to the respective diameters and are displayed as frequency distribution hitograms. Illustrative examples of measurements on uniform microspheres, cultured mammalian cells and human exfoliated gynecologic cells are presented.     

A convenient on-line device for reagent addition, sample mixing, and temperature control of cell suspensions in flow cytometry

We describe a simple and inexpensive device which permits the addition of up to three different solutions into a cell suspension which is on-line in a flow cytometer. The mixing chamber houses a disposable plastic cuvette stirred with a magnetic stirrer. The sample chamber is attached to a circulating water bath, hence accurate temperature control is achieved. Because the system is prepressurized and the sample line is very short, the delay time-between the point of sample modification and the point of analysis is reduced to a few seconds. Thus reagents may be added rapidly, and kinetic measurements of high temporal resolution are possible. Because the temperature of the sample chamber is regulated, binding can be observed over longer time periods than was previously possible. We demonstrate the usefulness of this device in determining the binding of fluoresceinated hexapeptide to human neutrophils under conditions where the stimulus is infused into the cell suspension while on-line in the cytometer.     

Utilization of Electrocardiographic P-wave Duration for AV Interval Optimization in Dual-Chamber Pacemakers

Background

Empiric programming of the atrio-ventricular (AV) delay is commonly performed during pacemaker implantation. Transmitral flow assessment by Doppler echocardiography can be used to find the optimal AV delay by Ritter''s method, but this cannot easily be performed during pacemaker implantation. We sought to determine a non-invasive surrogate for this assessment. Since electrocardiographic P-wave duration estimates atrial activation time, we hypothesized this measurement may provide a more appropriate basis for programming AV intervals.

Methods

A total of 19 patients were examined at the time of dual chamber pacemaker implantation, 13 (68%) being male with a mean age of 77. Each patient had the optimal AV interval determined by Ritter''s method. The P-wave duration was measured independently on electrocardiograms using MUSE® Cardiology Information System (version 7.1.1). The relationship between P-wave duration and the optimal AV interval was analyzed.

Results

The P-wave duration and optimal AV delay were related by a correlation coefficient of 0.815 and a correction factor of 1.26. The mean BMI was 27. The presence of hypertension, atrial fibrillation, and valvular heart disease was 13 (68%), 3 (16%), and 2 (11%) respectively. Mean echocardiographic parameters included an ejection fraction of 58%, left atrial index of 32 ml/m², and diastolic dysfunction grade 1 (out of 4).

Conclusions

In patients with dual chamber pacemakers in AV sequentially paced mode and normal EF, electrocardiographic P-wave duration correlates to the optimal AV delay by Ritter''s method by a factor of 1.26.     

In vitro fertilization with flow-cytometrically-sorted bovine sperm

An attractive feature of IVF is that fewer sexed sperm are needed than for artificial insemination. However, sperm sexed by flow cytometry/cell sorting are probably pre-capacitated, necessitating modifications to standard IVF systems for optimal success. With current procedures, the percentages of oocytes fertilized with sorted and unsorted frozen bovine sperm are similar, and events during the first cell cycle are timed similarly for sorted and unsorted sperm. However, in most cases, blastocyst production with sorted sperm was approximately 70% of controls produced with unsorted sperm. In some early studies, there appeared to be an unexplained delay of about half a day in blastocyst development. Nevertheless, some dozens of apparently normal calves, pre-sexed with 90% accuracy, have resulted from frozen embryos produced via IVF with sexed sperm. IVF also has proven useful as a bioassay for improving sperm-sorting procedures such as determining potential detrimental effects of laser power. It is likely that use of IVF in cattle breeding programs will increase considerably when sexed, frozen sperm become commercially available.     

Collection characteristics of a batch-type wetted wall bioaerosol sampling cyclone

The collection efficiency and sample retention of a batch-type wetted wall bioaerosol sampling cyclone (BWWC) were experimentally characterized. The BWWC is designed to sample air at 400 l/min and concentrate the particles into 12 ml of water. Aerosol is transported into a cylindrically-shaped axial flow cyclone through a tangential slot and the particles are impacted on the inner wall, which is wetted by air shear acting on a liquid pool at the base of the cyclone. The retention of collected particles and the aerosol collection efficiency of the BWWC were evaluated with polystyrene latex beads (PSL), sodium fluorescein/oleic acid droplets, and Bacillus atrophaeus (aka BG) spores. The retention of particles was determined by adding hydrosol directly into the device, running the BWWC for a pre-set period of time, and then determining the amount of particulate matter recovered relative to the initial amount. For 1-μm diameter PSL, 90% of the particles were recoverable from the cyclone body immediately after their introduction; however, only 10% were retained in the collection liquid after 8 h of operation. The aerosol sampling efficiency was determined by comparing the amount of particulate matter collected in the liquid with that collected by a reference filter. The collection efficiency was 50–60% for 1- and 3-μm polystyrene (PSL) particles, and 1.5% for 10-μm oleic acid particles. The efficiency for 3-μm oleic acid droplets was 35%. Explanations are provided for the difference between liquid and solid particle behavior, and for the low efficiency for the large liquid particles. The collection efficiency for single spore BG was slightly lower than that for 1-μm PSL.     

Flow cytometric purification of Alzheimer's disease amyloid plaque core protein using thioflavin T

Amyloid plaque core protein (APCP) of Alzheimer's disease obtained from brain tissue homogenate is difficult to recover in pure form, primarily because of contaminating lipofuscin (LF) granules. Thioflavin T, a fluorescent dye previously used to stain amyloid, was found to bind to APCP but not to lipofuscin. The latter, however, is autofluorescent. Fluorometric studies showed that at 370 nm excitation APCP has a maximal emission at 418 nm, whereas the autofluorescent LP has a maximal emission at 450 nm. This difference in emission permitted the use of a flow cytometer-sorter (FACS 440) for purification of APCP. APCP particles fluoresced distinctly from LF granules on the log blue fluorescence parameter. The two entities were sorted using forward light scatter versus fluorescence. A collection apparatus was designed and prepared to facilitate the collection of large volumes of sheath fluid and particles and to minimize fragmentation of particles during the collection process. The sorted APCP fraction was 98% pure. This work demonstrates how old dyes can be used to perform new tricks and provide a useful method for separating complex protein.     

A versatile chamber for simultaneous measurements of oxygen exchange and fluorescence in filamentous and thallous algae as well as higher plants

A new chamber was developed for a simultaneous measurement of fluorescence kinetics and oxygen exchange in filamentous and thallous algae as well as in small leaves of water plants. Algal filaments or thalli are kept by a stainless grid close to the bottom window of the chamber in the sample compartment. The grid separates the object from the electrode compartment with the oxygen electrode at the top. This compartment accommodates, in addition, a magnetic stirrer that provides efficient circulation of the medium between the sample and the electrode. This magnetic bar spins on a fixed axis and is driven by an electronically commutated magnetic field produced by four coils which are arranged around the chamber. This design yields a very favourable signal to noise ratio in the oxygen electrode records. Consequently, measurements can be performed even of algae with very low photosynthetic rates such as marine low-light red algae or algae under severe stress. For irradiation of the samples and for fluorescence measurements a fibre optic light guide is used facing the window of the chamber. The four branches of a commercially available light guide serve the following purposes: collection of sample fluorescence and supply of measuring, actinic, and saturating light, respectively.     

Control and optimization of apheresis procedures in a COBE 2997 cell separator

To obtain more efficient operation of a COBE Model 2997 clinical cell separator using either a Single Stage II (SS II) or a Dual Stage separation chamber, modifications were made to allow complete computer control. Product cell density was detected using an optical sensor and controlled by automatic feedback through a microcomputer interface. Control was accomplished by automatically adjusting the red blood cell (RBC) and plasma product flow rates using a proportional-integral (PI) algorithm. Results show that, using either chamber, the product cell density can be maintained at a preselected value for extended periods of time without operator intervention. This system allowed investigation of optimal operating regions for plateletpheresis and leukapheresis procedures. The effects of centrifuge rpm and controller set point on centrifuge operation were investigated using a second order factorial experimental design. Theoretical significance of model parameters was assessed with the aid of a hindered settling model and simple reasoning about the interface position relative to the collection port. The results suggest that, in either chamber, the optimum operating region for plateletpheresis procedures occurs at moderate controller set points and high centrifuge rpm. The resultant operating efficiency and product purity values are approximately 63 percent and 0.65 respectively in the SS II chamber and approximately 70 percent and 0.70 respectively in the Dual Chamber. In the SS II, the optimum operating region for leukapheresis procedures occurred at high controller set point values for any centrifuge rpm above 1200 with an operating efficiency near 100 percent. However, in the Dual Chamber, the optimum operating region for leukapheresis procedures occurred at high controller set points and high centrifuge rpm's, again providing an operating efficiency near 100 percent.     

Automated formation of multicomponent-encapuslating vesosomes using continuous flow microcentrifugation

Vesosomes – hierarchical assemblies consisting of membrane-bound vesicles of various scales – are potentially powerful models of cellular compartmentalization. Current methods of vesosome fabrication are labor intensive, and offer little control over the size and uniformity of the final product. In this article, we report the development of an automated vesosome formation platform using a microfluidic device and a continuous flow microcentrifuge. In the microfluidic device, water-in-oil droplets containing nanoscale vesicles in the water phase were formed using T-junction geometry, in which a lipid monolayer is formed at the oil/water interface. These water-in-oil droplets were then immediately transferred to the continuous flow microcentrifuge. When a water-in-oil droplet passed through a second lipid monolayer formed in the continuous flow microcentrifuge, a bilayer-encapsulated vesosome was created, which contained all of the contents of the aqueous phase encapsulated within the vesosome. Encapsulation of nanoscale liposomes within the outer vesosome membrane was confirmed by fluorescence microscopy. Laser diffraction analysis showed that the vesosomes we fabricated were uniform (coefficient of variation of 0.029). The yield of the continuous flow microcentrifuge is high, with over 60% of impinging water droplets being converted to vesosomes. Our system provides a fully automatable route for the generation of vesosomes encapsulating arbitrary contents. The method employed in this work is simple and can be readily applied to a variety of systems, providing a facile platform for fabricating multicomponent carriers and model cells.     

The sample processing time interval as an influential factor in flow cytometry analysis of lymphocyte subsets

The objective of this paper is to propose a protocol to analyze blood samples in yellow fever 17DD vaccinated which developed serious adverse events. We investigated whether or not the time between sample collection and sample processing could interfere in lymphocyte subset percentage, for it is often impossible to analyze blood samples immediately after collection due to transport delay from collection places to the flow cytometry facility. CD4+CD38+ T, CD8+CD38+ T, CD3+ T, CD19+ B lymphocyte subsets were analyzed by flow cytometry in nine healthy volunteers immediately after blood collection and after intervals of 24 and 48 h. The whole blood lysis method and gradient sedimentation by Histopaque were applied to isolate peripheral blood mononuclear cells for flow cytometry analyses. With the lysis method, there was no significant change in lymphocyte subset percentage between the two time intervals (24 and 48 h). In contrast, when blood samples were processed by Histopaque gradient sedimentation, time intervals for sample processing influenced the percentage in T lymphocyte subsets but not in B cells. From the results obtained, we could conclude that the whole blood lysis method is more appropriate than gradient sedimentation by Histopaque for immunophenotyping of blood samples collected after serious adverse events, due to less variation in the lymphocyte subset levels with respect to the time factor.     

Transport characteristics of expiratory droplets and droplet nuclei in indoor environments with different ventilation airflow patterns

Expiratory droplets and droplet nuclei can be pathogen carriers for airborne diseases. Their transport characteristics were studied in detail in two idealized floor-supply-type ventilation flow patterns: Unidirectional-upward and single-side-floor, using a multiphase numerical model. The model was validated by running interferometric Mie imaging experiments using test droplets with nonvolatile content, which formed droplet nuclei, ultimately, in a class-100 clean-room chamber. By comparing the droplet dispersion and removal characteristics with data of two other ceiling-supply ventilation systems collected from a previous work, deviations from the perfectly mixed ventilation condition were found to exist in various cases to different extent. The unidirectional-upward system was found to be more efficient in removing the smallest droplet nuclei (formed from 1.5 mum droplets) by air extraction, but it became less effective for larger droplets and droplet nuclei. Instead, the single-side-floor system was shown to be more favorable in removing these large droplets and droplet nuclei. In the single-side-floor system, the lateral overall dispersion coefficients for the small droplets and nuclei (initial size 相似文献