A low-voltage droplet charging circuit with simulative cell-sorting function for flow cytometer-cell sorter

BACKGROUND: Flow cytometer cell sorters have become important tools in many biological laboratories. Commercial electrically-deflected cell sorters that deflect wanted cells in electrically charged droplets need high-voltage amplifiers which are expensive and difficult to obtain. Effort was made to build an alternative droplet charging circuit with low-voltage amplifiers that are much easier to get and have more reasonable price. METHODS: A low-voltage charging circuit was designed. Every time a cell was to be separated, a pair of complementary charging pulses were produced: one was positive and the other was negative with equal amplitude. These were enlarged by two low-voltage charging amplifiers to drive two charging electrodes respectively. RESULTS: Due to the effect of addition, the voltage between the two electrodes was double as high as the output of either amplifier. The result of test experiment proved that the cell sorter with low-voltage amplifiers, which was cheaper and easier to obtain, could separate cells as efficiently as the instrument with high-voltage ones that were more expensive and more difficult to make. In addition, a simulative cell-sorting function was provided. CONCLUSIONS: This low-voltage, easily-built and low-price charging circuit for flow cytometer cell sorter is a good alternative to the commonly used high-voltage one, especially to researcher who hopes to build his own personal instrument.     

Enrichment during transdominant genetic experiments using a flow sorter

BACKGROUND: Flow cytometry, in combination with retroviral expression libraries, is a powerful tool for genetic experimentation in mammalian cells. Expression libraries are transduced into cells engineered with a fluorescent reporter. Sorting for either bright or dim cells allows enrichment for specific inhibitors that alter reporter activity. This strategy has been used to isolate peptides and RNAs that either activate or suppress defined biochemical pathways. METHODS: Several variables contribute to the enrichment process: (1) the background of the fluorescence bioassay; (2) the mean fluorescence ratio between the induced and noninduced reporter cell populations; (3) the genetic penetrance, or strength, of the inhibitor; and (4) the multiplicity of infection (MOI). An experimental and theoretical analysis, including computer modeling, of these issues in the context of a mammalian cell bioassay was undertaken. RESULTS: MOI measurements were shown to be problematic. High MOI had little effect on enrichment early in the cycling process but a significant effect at later stages. Penetrance and background were critical throughout the process. Enrichments within about twofold of the theoretical maximum were observed. CONCLUSIONS: Caution should be exercised in MOI determination because of the danger of significant underestimation. High MOI is potentially advantageous early in the selection process but hinders enrichment in the later rounds. Modeling shows that MOI, assay background and clone penetrance are the principal variables that determine the success of transdominant selections by FACS.     

A microfabricated fluorescence-activated cell sorter.

We have demonstrated a disposable microfabricated fluorescence-activated cell sorter (microFACS) for sorting various biological entities. Compared with conventional FACS machines, the microFACS provides higher sensitivity, no cross-contamination, and lower cost. We have used microFACS chips to obtain substantial enrichment of micron-sized fluorescent bead populations of differing colors. Furthermore, we have separated Escherichia coli cells expressing green fluorescent protein from a background of nonfluorescent E. coli cells and shown that the bacteria are viable after extraction from the sorting device. These sorters can function as stand-alone devices or as components of an integrated microanalytical chip.     

Two new isotype-specific switching activities detected for Ig class switching

Ig class switch recombination (CSR) occurs by an intrachromosomal deletional process between switch (S) regions in B cells. To facilitate the study of CSR, we derived a new B cell line, 1.B4.B6, which is uniquely capable of mu --> gamma3, mu --> epsilon, and mu --> alpha, but not mu --> gamma1 CSR at its endogenous loci. The 1.B4.B6 cell line was used in combination with plasmid-based isotype-specific S substrates in transient transfection assays to test for the presence of trans-acting switching activities. The 1.B4.B6 cell line supports mu --> gamma3, but not mu --> gamma1 recombination, on S substrates. In contrast, normal splenic B cells activated with LPS and IL-4 are capable of plasmid-based mu --> gamma1 CSR and demonstrate that this S plasmid is active. Activation-induced deaminase (AID) was used as a marker to identify existing B cell lines as possible candidates for supporting CSR. The M12 and A20 cell lines were identified as AID positive and, following activation with CD40L and other activators, were found to differentially support mu --> epsilon and mu --> alpha plasmid-based CSR. These studies provide evidence for two new switching activities for mu --> gamma1 and mu --> epsilon CSR, which are distinct from mu --> gamma3 and mu --> alpha switching activities previously described. AID is expressed in all the B cell lines capable of CSR, but cannot account for the isotype specificity defined by the S plasmid assay. These results are consistent with a model in which isotype-specific switching factors are either isotype-specific recombinases or DNA binding proteins with sequence specificity for S DNA.     

Sample flow switching techniques on microfluidic chips

This paper presents an experimental investigation into electrokinetically focused flow injection for bio-analytical applications. A novel microfluidic device for microfluidic sample handling is presented. The microfluidic chip is fabricated on glass substrates using conventional photolithographic and chemical etching processes and is bonded using a high-temperature fusion method. The proposed valve-less device is capable not only of directing a single sample flow to a specified output port, but also of driving multiple samples to separate outlet channels or even to a single outlet to facilitate sample mixing. The experimental results confirm that the sample flow can be electrokinetically pre-focused into a narrow stream and guided to the desired outlet port by means of a simple control voltage model. The microchip presented within this paper has considerable potential for use in a variety of applications, including high-throughput chemical analysis, cell fusion, fraction collection, sample mixing, and many other applications within the micro-total-analysis systems field.     

A simple electronic volume cell sorter for clonogenicity assays

A single-parameter electronic volume flow cell sorter that can be easily and inexpensively constructed using existing technology is described. The instrument is designed for ease and flexibility of operation, including such features as a large open area for recovering sorted cells into a variety of dishes or vessels; a remote, electrically activated fluidics system; a mechanism for heating or cooling samples during sorting; a simple arrangement for monitoring and adjusting the sorting control parameters; and an interface to a standard IBM personal computer for data acquisition, analysis, and control of the sorting windows. Several researchers in our laboratory now routinely use this sorter for plating precise numbers of cells directly into culture dishes in an aseptic manner for clonogenicity assays. The instrument can sort cells at rates of up to approximately 2,000 per second with greater than 80% sorting efficiency and no cytotoxicity. An advantage of this system is that the sorting windows can be set to exclude acellular debris and include either the entire cell volume distribution or a subset thereof. Applications of the instrument are detailed, including 1) precise cell plating for low-dose survival studies, 2) separation of cells into age compartments, and 3) rapid inoculation of single cells into multiwell dishes for cloning studies. Advantages of this technology for cell survival studies are detailed, along with some limitations to its applicability.     

A note on the poiseuille-type flow of a casson fluid

When a Casson fluid flows in a tube, there is a central region of plug flow. It is shown that, unless hematocrit varies markedly with radius, this region is so small that it may be neglected in all physiologically relevant cases.     

A new method of sampling ascitic fluid from rats

A new method of sampling ascitic fluid from rats over a period of at least 8 h in a reliable and easy way is described. The objective was to determine drug concentrations in ascitic fluid after intraperitoneal chemotherapy. Two silicon tubes were implanted into the abdominal cavity, one for drug administration and regulation of pressure, the other enabled ascitic fluid to be withdrawn.     

A new principle of continuous flow cell cultivation

Summary A completely liquid-filled culture chamber with gas exchange across a synthetic membrane (Larsen andNilsson 1985) was incorporated into an automatic continuous flow system. The absence of an airliquid interface in the system permits removal of cell samples, and addition of fresh medium, under strictly sterile conditions. In this system,Tetrahymena pyriformis can be kept under optimal growth conditions in a rich nutrient medium and any defined cell density may be maintained for extended periods of time by varying the dilution rate of the culture. Furthermore, it has been possible to demonstrate, in the slope of the growth curve, even small changes which are difficult to detect in batch cultures since the duration of these changes is short. In the continuous flow system, the relative cell volume distribution and the food vacuole forming capacity of the cells were unaltered; however, all cells contained small refractive granules. The system permits the culture volume to be varied, but a standard volume of 20 ml was maintained in most experiments. Since the culture volume is small, the system requires less than one liter of fresh medium per week to maintain the cells in the exponentially multiplying growth phase.     

The Heidelberg flow analyzer and sorter (HEIFAS) approach on the prescreening of uterine cancer.

A dual laser flow system has been proved and used with a novel staining method for simultaneous quantitative DNA and protein analysis. A diamidinophenylindole compound (DAPI) for DNA has been employed in combination with sulforhodamine (SR 101) for protein. With this dye mixture various cell types of the cervical smear could be identified. Despite some overlapping, clusters of leukocytes, endocervicals, (para)basals, intermediates, superficials and dys/neoplastic cells together with artificial events could be discriminated. Up to now the problem of negative cells which create positive signals (false alarms) in the dys/neoplastic region could not be sufficiently solved.     

A new flow co-culture system for studying mechanobiology effects of pulse flow waves

Artery stiffening is known as an important pathological change that precedes small vessel dysfunction, but underlying cellular mechanisms are still elusive. This paper reports the development of a flow co-culture system that imposes a range of arterial-like pulse flow waves, with similar mean flow rate but varied pulsatility controlled by upstream stiffness, onto a 3-D endothelial-smooth muscle cell co-culture. Computational fluid dynamics results identified a uniform flow area critical for cell mechanobiology studies. For validation, experimentally measured flow profiles were compared to computationally simulated flow profiles, which revealed percentage difference in the maximum flow to be <10, <5, or <1% for a high, medium, or low pulse flow wave, respectively. This comparison indicated that the computational model accurately demonstrated experimental conditions. The results from endothelial expression of proinflammatory genes and from determination of proliferating smooth muscle cell percentage both showed that cell activities did not vary within the identified uniform flow region, but were upregulated by high pulse flow compared to steady flow. The flow system developed and characterized here provides an important tool to enhance the understanding of vascular cell remodeling under flow environments regulated by stiffening.

Electronic supplementary material

The online version of this article (doi:10.1007/s10616-012-9445-2) contains supplementary material, which is available to authorized users.     

A new technique for monitoring pollen flow in orchids

Summary The orchid Prasophyllum fimbria is pollinated by nectar-feeding native bees and wasps. The pollinia are patially separated from the viscidium by a stipe so that pollinia can be labelled with coloured histochemical stains without interfering with pollinarium removal. Pollen flow was monitored by following the movement of the coloured pollen in several populations of P. fimbria in Western Australia. Statistical analysis confirmed that pollen labelling did not interfere with pollinarium removal or subsequent pollination of the labelled flower. Fifty eight labelled pollinaria were removed by vectors from 16 test spikes, with a total of 125 flowers on 47 spikes receiving labelled pollen. An average of 2 flowers received pollen for every pollinium removed but up to 6 flowers received pollen from a single collinium. No significant differences between mean vector flights and pollen flow distances were detected. On average, geitonogamous transfers only accounted for 22% of all pollinations. This is a simple and inexpensive technique for the direct labelling of pollen with minimal disruption to the pollination system and may have applications in other plant families.     

The structure of an endosomal protein sorter

Three "endosomal sorting complexes required for transport," ESCRT-I, -II, and -III, mediate sorting of ubiquitinated membrane proteins into intraluminal endosomal vesicles that are destined for degradation in lysosomes. Two recent reports, one in Nature and one in this issue of Developmental Cell, reveal the crystal structure of the yeast form of ESCRT-II.     

A novel solution for fluid flow problems based on the lattice Boltzmann method

Recently, phase separation and fluid flow problems have represented an important development in fluid dynamics, which has many important industrial applications. Lattice Boltzmann method (LBM) is the numerical method that explains the behaviour of fluid dynamics in mesoscopic scale single-component single-phase and multi-component multiphase flows. In this paper, we study the lattice Boltzmann models (LBMs) in two dimensions (2D) with nine directions (Q9), that is the D2Q9 model was used to study the phase separation and observe that the phenomenon of fluid flow in a cylinder has obstacle and square cavity. The simulation results show that fluid flows in the square cavity and in the cylinder, present phase separation of single-component multiphase fluid flow.     

A new method to predict flowability using a microscale fluid bed

The purpose of this research was to develop a new method to predict the flow behavior of pharmaceutical powders using a multichamber microscale fluid bed. Different amounts of poorly flowing paracetamol were added to various grades of microcrystalline celluloses and silicified microcrystalline cellulose powders. Magnesium stearate was used as a lubricant. Experimental minimum fluidization velocities (u _mf) were defined using 2 to 4 g (equal to 10 mL) of material (Video 1). The reference flowability of the powders was determined using a specific flow meter. Also, the weight variation of the compressed powders, using a single-punch press, was measured. When the amount of paracetamol in the excipients was increased, the experimentalu _mf increased and the fluidization behavior grew worse (Video 2). Principal component analysis (PCA) established that the pressure difference over the bed as a function of fluidization velocity could be used to characterize the behavior of powders. The increase in poor fluidization behavior of the powders was in accordance with the increasing amount of paracetamol and with the increasing weight variation of the tablets. Furthermore, the angle of repose and the flow rate of silicified microcrystalline cellulose powders were predicted using a partial least squares (PLS) model. The developed method to predict flowability is a promising approach for use in the preformulation and formulation stages of new drug candidates, for example.