Conversion of logarithmic channel numbers into relative linear fluorescence intensity

We describe a simple, reproducible, and generally applicable method to assess the performance of log amplifiers by using a fluorescent sample that provides multiple peaks of different intensities. The channel differences between multiple peaks are used to evaluate the logarithmic behavior of the fluorescence signal amplifier on the flow cytometer. A calibration curve can be created to correct the channel numbers for deviations from true logarithmic behavior and then convert data into relative linear intensities. By using these linear fluorescent intensities, we compared the capacity of different antisera against HIV-1 (human immunodeficiency virus type 1) peptides to inhibit the binding of HIV-1 to CEM, a CD4-positive T-cell line. A wide range of applications for this calibration procedure can be envisioned and the method is valuable for monitoring instrument performance over time.     

Calibration of a flow cytometer against a microphotometer for morphologic cell identification

The calibration of a flow cytometer against a microphotometer, to allow the correlation of cell morphology with fluorescence intensity, is described. Using three human lymphoblastoid cell lines, the photomultiplier amplification of the microphotometer and the flow cytometer that gave optimum linearity between fluorescence intensity and DNA content for the two instruments was established. Thereafter, at these settings, there was satisfactory linear agreement between the fluorescence intensity profiles, as measured by the flow cytometer and the microphotometer, of stained cell populations. Day-to-day variation was also minimal, and it was demonstrated that the application of this procedure can provide an alternative to the employment of the sorting facility of a flow cytometer for the morphologic identification of cell subpopulations during flow cytometric analysis.     

DNA signal splitting improves detection and analysis of tetraploid populations.

Detection of DNA tetraploid populations requires a high index of suspicion at the time of data acquisition and frequently requires subsequent off-line analysis for confirmation, including evaluation of the hypertetraploid region. To analyze these specimens, the flow cytometer operator must run all specimens with the G0G1 peak in low channels or rerun specimens in which tetraploidy is suspected with a lower photomultiplier tube (PMT) voltage or lower amplifier gain setting. Re-analysis may not be possible in specimens with few cells. A simple modification to the cytometer allows PMT signal splitting with simultaneous processing of the signal by two different amplifiers. This allows simultaneous acquisition of histograms optimized for both the hypotetraploid and hypertetraploid regions.     

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.     

Modified Box-Cox transform for modulating the dynamic range of flow cytometry data

We describe an algorithm, Vout = Integer ([2(12)-1/2(12 lambda)-1] V lambda in-1) + 1; lambda greater than 0 based upon Box-Cox transformations as an alternative to nonlinear electronic amplifiers to expand or compress high- or low-amplitude flow cytometer-derived signals. If the indexing parameter lambda less than 1, input channels in the high-amplitude input range are compressed in the output range as occurs when an electronic logarithmic amplifier is used. However, if lambda greater than 1, input channels in the low-amplitude input range are compressed in the output range as occurs when an electronic power amplifier is used. Our modified Box-Cox transform can be implemented either during data collection or off-line for the transformation of previously collected raw data. The transform is the equivalent of an infinite class of nonlinear amplifiers. As the transform is implemented in software, it does not suffer from many of the disadvantages of nonlinear electronic amplifiers.     

Is there a role for amplifiers in sexual selection?

The amplifier hypothesis states that selection could favour the evolution of traits in signallers that improve the ability of receivers to extract honest information from other signals or cues. We provide a formal definition of amplifiers based on the receiver's mechanisms of signal perception and we present a game-theoretical model in which males advertise their quality and females use sequential-sampling tactics to choose among prospective mates. The main effect of an amplifier on the female mating strategy is to increase her mating threshold, making the female more selective as the effectiveness of the amplifier increases. The effects of the amplifier on male advertising strategy depends both on the context and on the types of the amplifier involved. We consider two different contexts for the evolution of amplifiers (when the effect of amplifiers is on signals and when it is on cues) and two types of amplifiers (the ‘neutral amplifier’, when it improves quality assessment without altering male attractiveness, and the ‘attractive amplifier’, when it improves both quality assessment and male attractiveness). The game-theoretical model provides two main results. First, neutral and attractive amplifiers represent, respectively, a conditional and an unconditional signalling strategy. In fact, at the equilibrium, neutral amplifiers are displayed only by males whose advertising level lays above the female acceptance threshold, whereas attractive amplifiers are displayed by all signalling males, independent of their quality. Second, amplifiers of signals increase the differences in advertising levels between amplifying and not-amplifying males, but they decrease the differences within each group, so that the system converges towards an ‘all-or-nothing’ signalling strategy. By applying concepts from information theory, we show that the increase in information transfer at the perception level due to the amplifier of signals is contrasted by a decrease in information transfer at the emitter level due to the increased stereotypy of male advertising strategy.     

ＧＭ（１，Ｎ）模型对生物系统应用的研究

灰色系统理论的GM(1,N)模型已被广泛地用于生态,农业、林业等与生命现象有关的系统的分析和处理。但GM(1,N)本质上是一线性系统,在生态系统中,由于个体和种群的发展均受个体和种群间对有限资源竞争的限制,一般地说线性假设不能成立。本文对GM(1、N)在生态系统的适应性进行了探讨,提出丁系统线性度,系统非线性显著度的概念、定义和具体计算方法,并在此基础上建立了GM(1,N)模型的适用标准。将这一标准施用于计算机模拟的非线性程度不同的生态系统,结果说明本文所提出的系统表征的度量标准基本准确。另外,本文对GM(1,N)中的参数拟合略有改进,新方法在计算上有合理。省时等特点。     

Time interval gating for analysis of cell function using flow cytometry.

We propose a method which significantly shortens the time required for both the collection and analysis of data derived from multiple sample, flow cytometric kinetic assays. We have defined the term Time Interval Gating (TIG) to describe this method. TIG effectively allows one flow cytometer to concurrently monitor several samples over the course of a kinetic assay. Data for all samples are stored in a single FCS 2.0 compatible listmode data file which we refer to as the TIG data file. TIG is adaptable to most commerical flow cytometers. Standard listmode analysis software can be used to analyze the TIG data files and correlate any combination of tubes and/or time intervals from the assay. Results for the entire assay can be displayed on a single two parameter plot. This paper describes how TIG is applied to neutrophil oxidative burst measurement using a standard EPICS Elite flow cytometer. In this assay, 11 samples were each monitored for 30 min to identify the extent to which volatile organic chemicals (VOCs) inhibited the oxidation of DCFH in stimulated neutrophils. TIG makes the oxidative burst assay practical for high volume screening by reducing the overall flow cytometer and analysis time required by a factor of ten. In addition, TIG provides an organized approach to managing data acquisition on instruments equipped with automated sampling systems.     

The allometry between secondary sexual traits and body size is nonlinear among cervids

Allometric relationships between sexually selected traits and body size have been extensively studied in recent decades. While sexually selected traits generally display positive allometry, a few recent reports have suggested that allometric relationships are not always linear. In male cervids, having both long antlers and large size provides benefits in terms of increased mating success. However, such attributes are costly to grow and maintain, and these costs might constrain antler length from increasing at the same rate as body mass in larger species if the quantity of energy that males can extract from their environment is limiting. We tested for possible nonlinearity in the relationship between antler size and body mass (on a log–log scale) among 31 cervids and found clear deviation from linearity in the allometry of antler length. Antler length increased linearly until a male body mass threshold at approximately 110 kg. Beyond this threshold, antler length did not change with increasing mass. We discuss this evidence of nonlinear allometry in the light of life-history theory and stress the importance of testing for nonlinearity when studying allometric relationships.     

Influence of Plasma Unsteadiness on the Spectrum and Shape of Microwave Pulses in a Plasma Relativistic Microwave Amplifier

Dependence of the shape of a microwave pulse in a plasma relativistic microwave amplifier (PRMA) on the initial plasma electron density in the system is detected experimentally. Depending on the plasma density, fast disruption of amplification, stable operation of the amplifier during the relativistic electron beam (REB) pulse, and its delayed actuation can take place. A reduction in the output signal frequency relative to the input frequency is observed experimentally. The change in the shape of the microwave signal and the reduction in its frequency are explained by a decrease in the plasma density in the system. The dynamics of the plasma density during the REB pulse is determined qualitatively from the experimental data by using the linear theory of a PRMA with a thin-wall hollow electron beam. The processes in a PRMA are analyzed by means of the KARAT particle-in-cell code. It is shown that REB injection is accompanied by an increase in the mean energy of plasma electrons and a significant decrease in their density.     

Systematic error is of minor importance to feedback structure estimates derived from time series of nonlinear population indices

Most modern population dynamics analyses of time series use simple population indices for ecological inference. These indices, collected for many years for various agricultural pests or game animals, are generally believed not to distort systematically feedback estimates because the assumption of linearity to population size roughly holds. To assess the relative importance of this assumption, we examined the effect of nonlinearity in a burrow index for voles on feedback estimates obtained through autoregressive modeling. We show that the issue of linearity is of less importance to ecological inference because the feedback estimates are routinely obtained on a logarithmic scale. Transforming data to logs has a strong linearization effect, removing most of the nonlinearity observed on the original scale. We conclude that the statistical tools for ecological inference, such as autoregressive log-linear models, are sufficiently robust to the systematic error imposed by index nonlinearity and that indices are valuable sources of ecological information even in situations when the assumed linear functional forms to population size were not exactly validated. We suggest that for time series modelers, the issue of a large sampling variation in small “noisy” populations is by far a more burning one than the systematic error due to index nonlinearity.     

Pressure and flow in the systemic arterial system

The dynamic characteristics of the proximal arterial system are studied by solving the nonlinear momentum and mass conservation equations for pressure and flow. The equations are solved for a model systemic arterial system that includes the aorta, common iliacs, and the internal and external iliac arteries. The model includes geometric and elastic taper of the aorta, nonlinearly elastic arteries, side flows, and a complex distal impedance. The model pressure wave shape, inlet and outlet impedance, wave travel, and apparent wave velocity compare favorably with the values measured on humans. Calculations indicate that: (i) reflections are the major factor determining the shape and distal amplification of the pressure wave in the arterial tree; (ii) although important in attenuating the proximal transmission of reflecting waves, geometric taper is not the major cause of the distal pressure wave amplification; (iii) the dicrotic wave is a result of peripheral reflection and is not due to the sudden change in flow at the end of systole; (iv) the elastic taper and nonlinearity of the wall elasticity are of minor significance in determining the flow and pressure profiles; and (v) in spite of numerous nonlinearities, the system behaves in a somewhat linear fashion for the lower frequency components.     

Plug flow cytometry extends analytical capabilities in cell adhesion and receptor pharmacology

BACKGROUND: Plug flow cytometry is a recently developed system for the automated delivery of multiple small boluses or "plugs" of cells or particles to the flow cytometer for analysis. Important system features are that sample plugs are of precisely defined volume and that the sample vessel need not be pressurized. We describe how these features enable direct cell concentration determinations and novel ways to integrate flow cytometers with other analytical instruments. METHODS: Adhesion assays employed human polymorphonuclear neutrophils (PMNs) loaded with Fura Red and Chinese hamster ovary (CHO) cells cotransfected with genes for green fluorescent protein (GFP) and human P-selectin. U937 cells expressing the human 7-transmembrane formyl peptide receptor were loaded with the fluorescent probe indo-1 for intracellular ionized calcium determinations. A computer-controlled syringe or peristaltic pump loaded the sample into a sample loop of the plug flow coupler, a reciprocating eight-port valve. When the valve position was switched, the plug of sample in the sample loop was transported to the flow cytometer by a pressure-driven fluid line. RESULTS: In stirred mixtures of PMNs and CHO cells, we used plug flow cytometry to directly quantify changes in concentrations of nonadherent singlet PMNs. This approach enabled accurate quantification of adherent PMNs in multicell aggregates. We constructed a novel plug flow interface between the flow cytometer and a cone-plate viscometer to enable real-time flow cytometric analysis of cell-cell adhesion under conditions of uniform shear. The High Throughput Pharmacology System (HTPS) is an instrument used for automated programming of complex pharmacological cell treatment protocols. It was interfaced via the plug flow coupling device to enable rapid (< 5 min) flow cytometric characterization of the intracellular calcium dose-response profile of U937 cells to formyl peptide. CONCLUSIONS: By facilitating the coupling of flow cytometers to other fluidics-based analytical instruments, plug flow cytometry has extended analytical capabilities in cell adhesion and pharmacological characterization of receptor-ligand interactions.     

Detection of mRNA in flow-sorted cells

Cells were sorted onto nitrocellulose filters which were saturated with a lysing cocktail designed to preferentially immobilize cellular mRNA. After washing, these filters were incubated with 32P-labeled specific DNA probes. We used the phorbol ester/lipopolysaccharide (PMA + LPS) co-induction of IL-1 mRNA and CD13 expression in U937 cells to demonstrate the specificity of the technique. In addition we used the abundant expression of c-fos in U937 to demonstrate linearity. IL-1 beta mRNA is readily discernable autoradiographically from as few as 5,000 PMA + LPS-induced cells sorted onto a filter. With liquid scintillation counting we demonstrate good linearity of the c-fos quantitation over the range of 1,000 cells to 60,000 cells per filter target. The technique is easily adapted to any sorting flow cytometer and should prove useful to help correlate any flow cytometric cell phenotype with specific mRNA abundance.     

Nonlinear principal components analysis of neuronal spike train data

Many recent approaches to decoding neural spike trains depend critically on the assumption that for low-pass filtered spike trains, the temporal structure is optimally represented by a small number of linear projections onto the data. We therefore tested this assumption of linearity by comparing a linear factor analysis technique (principal components analysis) with a nonlinear neural network based method. It is first shown that the nonlinear technique can reliably identify a neuronally plausible nonlinearity in synthetic spike trains. However, when applied to the outputs from primary visual cortical neurons, this method shows no evidence for significant temporal nonlinearities. The implications of this are discussed. Received: 29 November 1996 / Accepted in revised form: 1 July 1997     

Reliable amplification method for bacterial RNA

DNA microarray technology has been increasingly applied for studies of clinical samples. Frequently, RNA probes from clinical samples are available in limited amounts. We describe a reliable amplification method for bacterial RNA. We verified this method on mycobacterial RNA applying mycobacterial genome-directed primers (mtGDPs). Glass slide-based oligoarrays were employed to assess the quality of the amplification method. We observed a relatively small bias in amplified RNA pool when compared to the unamplified one. Up to 1000-fold linear RNA amplification in a single amplification round was obtained. To our knowledge, this study describes the first amplification method for mycobacterial RNA.     

RT-PCR法同时测定APP与tau mRNA的含量(英文)

β—淀粉样蛋白前体(APP)与tau蛋白是两种与Alzheimer病的病理改变相关的蛋白质,近来也发现它们或其修饰产物在包含体肌炎及慢性氯喹中毒的肌细胞中沉积。为更好地研究这两种相关蛋白质在肌细胞中的表达,我们建立了用逆转录-多聚酶链反应同时定量这两种基因转录产物的方法。本法选用甘油醛-3-磷酸脱氢酶(G_3PD)作为内标,用激光图像分析测定扩增产物。结果显示,PCR在很宽的范围内呈指数扩增,两种靶mRNA与内标mRNA扩增效率相当,因而可以G_3PD来校正APP与tau的测定结果。初步实验显示氯喹中毒首先促进肌肉细胞中APP与tau蛋白的表达,在第4周达到高峰,然后表达逐渐减少,在第10周转为明显抑制作用。     

Linear correlation between average fluorescence intensity of green fluorescent protein and the multiplicity of infection of recombinant adenovirus

Background

Adenoviral vector is an efficient tool for gene transfer. Protein expression is regulated by a number of factors, but the regulation by gene copy number remains to be investigated further.

Results

Assessed by flow cytometry, we demonstrated a significant linear correlation between average fluorescence intensity of green fluorescent protein (GFP) and a wide range of multiplicity of infection (MOI), spanning from 0.01 to 200. Average GFP intensity was calculated by mean fluorescence intensity (MFI) × percentage of infection (POI) (MFI × POI) and the correlation was observed in cells transduced with GFP-expressing adenoviral vector driven either by a cytomegalovirus (CMV) promoter for 3 to 6 h or by a human phosphoglycerate kinase (PGK) promoter for 18 to 24 h. Factors impacting this linear correlation include MOI of viral vector, strength of promoter driving GFP expression, cell type transduced and incubation time after gene transfer. We also found that weak GFP signals could be interfered by background signals, whereas strong GFP signals could overshot the detection limitation of the flow cytometer and resulted in a deviation from linearity which was prevented by adjusting the setting in flow cytometer. Moreover, we compared promoter strength as measured by MFI × POI and found that the relative activity of CMV promoter to PGK promoter was 20 to 47 folds in A549 cells and 32 to > 100 folds in H1299 cells.

Conclusions

The linear correlation between MFI × POI and a wide range of adenoviral MOI provides an efficient method to investigate factors regulating protein expression and to estimate virus titers.

Electronic supplementary material

The online version of this article (doi:10.1186/s12929-015-0137-z) contains supplementary material, which is available to authorized users.     

RT—PCR法同时测定APP与tau mRNA的含量

β－淀粉样蛋白前体（ＡＰＰ）与ｔａｕ蛋白是两种与Ａｌｚｈｅｉｍｅｒ病的病理改变相关的蛋白质,近年来也发现它们或其修饰产物在包含体肌炎及慢性氯喹中毒的肌细胞中沉积。为更好地研究这两种相关蛋白质在肌细胞中的表达,我们建立了用逆转录－多聚酶链反应同时定量这两种相关蛋白质在肌细胞中的表达,我们建立了用逆转录－多聚酶链反应同时定量这两种基因转录产物的方法。本法选用甘油醛－３－磷酸脱氢酶（Ｇ３ＰＤ）作为内标,     

Half-Octave Shift in Mammalian Hearing Is an Epiphenomenon of the Cochlear Amplifier

The cochlear amplifier is a hypothesized positive feedback process responsible for our exquisite hearing sensitivity. Experimental evidence for or against the positive feedback hypothesis is still lacking. Here we apply linear control theory to determine the open-loop gain and the closed-loop sensitivity of the cochlear amplifier from available measurements of basilar membrane vibration in sensitive mammalian cochleae. We show that the frequency of peak closed-loop sensitivity is independent of the stimulus level and close to the characteristic frequency. This implies that the half-octave shift in mammalian hearing is an epiphenomenon of the cochlear amplifier. The open-loop gain is consistent with positive feedback and suggests that the high-frequency cut-off of the outer hair cell transmembrane potential in vivo may be necessary for cochlear amplification.