A method of staining basal-cell plasma membranes in epidermal sheets

An adenosine triphosphatase method was devised to stain basal epidermal cell plasma membranes in sheet preparations of humans, rat, mouse and guinea pig epidermis. The method is useful for direct observation of sizes, shapes, and numbers of basal epidermal cells.     

Estimating the Median Generation Time of Proliferating Cell Systems in Steady State

For proliferating cell systems in which the usual "labeled mitoses" method cannot be used to estimate generation times, an alternative scheme is derived. The method presented here is based on observation (by autoradiography) of the median grain count of labeled interphase cells following a pulse of labeled DNA precursor. It is shown that the median generation time of the labeled cells will be equal to the time required for the median grain count to halve, starting from the time when half the labeled cells have completed their first division. This starting time is determined from observation of the first wave of labeled mitoses. The procedure was designed to minimize error resulting from such factors as radiation damage, label reutilization, and the use of a nonzero grain counting threshold. The method is applied to the analysis of two cases of acute leukemia in man.     

介绍一种兔VX2肿瘤的传代和接种方法及其应用经验和体会

目的:介绍一种兔VX2肿瘤的传代和接种方法及其应用经验和体会,从而更好的利用此模型进行生物医学研究。方法:从荷瘤新西兰大白兔取活性良好的肿瘤组织块,制备肿瘤细胞悬液,过滤后接种于健康成年新西兰兔左后肢肌肉内。通过一般观察、MRI和大体及病理学切片对肿瘤进行验证。结果:肿瘤传代和接种后生长良好,MRI、大体及组织学验证保持了VX2的肿瘤特点。结论:本研究介绍的兔VX2肿瘤的传代与接种方法稳定、可靠,值得大家推广和应用。     

A Rapid Experimental Method to Study Primary Embryonic Induction

The technique described here is a combination of the sandwich-method and cell culture. It allows one to know quickly whether induction occurred or not (48 h if cell spreading is considered, 4–5 days if morphological differentiation is observed). The dissociation of the explants soon after induction does not disturb their inductive pattern. Moreover, this method allows a daily observation of the morphological events and study at the cellular and/or molecular level.     

Pattern prediction for moving cells

Summary A continuous-time Markov chain model consisting of four positional and directional states is used to predict the eventual relative positioning of two motile cell types. It is assumed that the period of observation is small in comparison with the generation time of both cell types. The method is useful in predicting developmental phenomena and is applicable to complex patterns involving more than two types of cells.This research was supported by a grant from the National Research Council of Canada. Grant No. A 9072.     

Imaging Through the Pupal Case of Drosophila melanogaster

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled analysis of cell and developmental biology from a variety of methodological angles. Live imaging is an emerging method for observing dynamic cell processes, such as cell division or cell motility. Having isolated mutations in uncharacterized putative cell cycle proteins it became essential to observe mitosis in situ using live imaging. Most live imaging studies in Drosophila have focused on the embryonic stages that are accessible to manipulation and observation because of their small size and optical clarity. However, in these stages the cell cycle is unusual in that it lacks one or both of the gap phases. By contrast, cells of the pupal wing of Drosophila have a typical cell cycle and undergo a period of rapid mitosis spanning about 20 hr of pupal development. It is easy to identify and isolate pupae of the appropriate stage to catch mitosis in situ. Mounting intact pupae provided the best combination of tractability and durability during imaging, allowing experiments to run for several hours with minimal impact on cell and animal viability. The method allows observation of features as small as, or smaller than, fly chromosomes. Adjustment of microscope settings and the details of mounting, allowed extension of the preparation to visualize membrane dynamics of adjacent cells and fluorescently labeled proteins such as tubulin. This method works for all tested fluorescent proteins and can capture submicron scale features over a variety of time scales. While limited to the outer 20 µm of the pupa with a conventional confocal microscope, this approach to observing protein and cellular dynamics in pupal tissues in vivo may be generally useful in the study of cell and developmental biology in these tissues.     

Area-based analyzing technique at cell array experiment using neuronal cell line

We propose a simple procedure for the identification and quantitative analysis of neurite outgrowth in neuronal cell lines that were uniformly differentiated. Upon stimulation most neuronal cell lines extend neurites in the differentiation process, resulting, according to our observation, in the increase of cell surface area. This increase is dependent on the length and the number of extended neurites. Furthermore, we use this method for the phenotype analysis of cell array experiments to perform large-scale functional evaluation of genes involved in the neurite outgrowth during neuronal differentiation.     

A method to monitor replication fork progression in mammalian cells: nucleotide excision repair enhances and homologous recombination delays elongation along damaged DNA

The capacity to rescue stalled replication forks (RFs) is important for the maintenance of cell viability and genome integrity. Here, we have developed a novel method for monitoring RF progression and the influence of DNA lesions on this process. The method is based on the principle that each RF is expected to be associated with a pair of single-stranded ends, which can be analyzed by employing strand separation in alkali. This method was applied to examine the rate of RF progression in Chinese hamster cell lines deficient in ERCC1, which is involved in nucleotide excision repair (NER), or in XRCC3, which participates in homologous recombination repair, following irradiation with ultraviolet (UV) light or exposure to benzo(a)pyrene-7,8-diol-9,10-epoxide (BPDE). The endpoints observed were cell survival, NER activity, formation of double-strand breaks and the rate of RF progression. Subsequently, we attempted to explain our observation that cells deficient in XRCC3 (irs1SF) exhibit enhanced sensitivity to UV radiation and BPDE. irs1SF cells demonstrated a capacity for NER that was comparable with wild-type AA8 cells, but the rate of RF progression was even higher than that for the wild-type AA8 cells. As expected, cells deficient in ERCC1 (UV4) showed no NER activity and were hypersensitive to both UV radiation and BPDE. The observation that cells deficient in NER displayed a pronounced delay in RF progression indicates that NER plays an important role in maintaining fork progression along damaged DNA. The elevated rate of RF progression in XRCC3-deficient cells indicates that this protein is involved in a time-consuming process which resolves stalled RFs.     

儿童朗格汉斯细胞组织细胞增生症临床研究

目的:探讨观察儿童朗格汉斯细胞组织增生症临床治疗方案和效果。方法:选取我院2007年4月-2014年11月收治的儿童朗格汉斯细胞组织细胞增生症65例,按随机数字表法分为观察组(32例)和对照组(33例)。对照组给予朗格汉斯细胞组织细胞增生症-Ⅲ(LCH-Ⅲ)治疗方案,观察组给予难治性2008方案。观察两组患者临床疗效、复发率、并发症、生存率。结果:观察组完全缓解率显著高于对照组(P0.05),而两组患者的复发率和总有效率之间的差异无统计学意义(P0.05);两组治疗后9个月、12个月、24个月生存率差异无统计学意义(P0.05);观察组不良反应发生率为9.38%,对照组为24.24%,观察组稍低于对照组,但两组差异无统计学意义(P0.05)。结论:采用难治性2008方案治疗儿童朗格汉斯细胞组织细胞增生症较LCH-Ⅲ方案疗效更佳,且远期生存率明显改善,还可减少不良反应,值得在临床治疗中推广应用。     

昆明白小鼠胚胎干细胞分离与体外培养

为探索昆明白小鼠胚胎干细胞建系方法,将受孕4.5天的昆明白小鼠囊胚用免疫手术法去除滋胚层,然后将内细胞团(ICM)接种于胎鼠成纤维细胞饲养层上培养,形成的胚胎干细胞样集落用胰蛋白酶-EDTA消化法传代,培养后进行相差显微镜观察及碱性磷酸酶染色。结果饲养层上生长的ICM细胞呈典型的ES样细胞集落,传至第8代碱性磷酸酶染色呈强阳性。实验表明免疫手术法适用于昆明白小鼠ES细胞建系,获得的细胞集落具有ES细胞的主要生物学性状。     

Label-free observation of three-dimensional morphology change of a single PC12 cell by digital holographic microscopy

Observation of three-dimensional (3D) morphology changes of a single mammalian cell is very useful to understand cell response for various stimuli. Conventional techniques to evaluate morphology changes with sufficient precision and high temporal resolution are limited. For example, the confocal fluorescence microscope is available to take 3D morphology changes, whereas fluorescence microscopic observation requires labeling the cells with fluorescence dye. Recently, a novel imaging method based on digital holography was developed for nonlabeling microscopic observation of 3D morphology. Digital holographic microscopy has high potentiality in digital focusing properties, video-frequency capability, noninvasive operation, and so forth. It obtains a quantitative phase image of a living cell from a single recorded hologram, with interferometric accuracy, and surveys the rapid morphology change of a single cell. In this study, digital holographic microscopy was applied to monitor the 3D morphology change of an individual PC12 cell, a nerve model cell, subjected to high K(+) stimulation. Phase images of the rapidly swelling cell were acquired, and time lapse reconstruction of 3D cell morphology was performed from phase images. Our results demonstrate that digital holographic imaging is a powerful new tool for evaluation of cell response against various stimulants without any labeling reagent.     

Novel antigen system for tracking epithelial cell migration in serum-free culture

We report here a unique system for tracking normal human urothelial cell migration in serum-free culture medium (HMRI-1). The key observation was that urothelial cells deposited red blood cell surface antigen on the culture dish in a remarkable pattern. Scrutiny of this pattern showed that each migrating cell left behind antigen imprints which formed parallel tracks the width of the cell. Hence the previous migratory history of the cells was instantly mapped by simply visualizing the antigen tracks deposited by the cells on the dish. Apart from providing a simple method for tracking urothelial cells, this observation has wider implications for mechanistic studies of epithelial cell movement in general. It also highlights the complicating effects associated with the addition of serum as a traditional culture supplement, since the inclusion of serum in the HMRI-1 medium abolished the above effect by inhibiting cell migration.     

基于微流控的真菌单细胞捕获和培养

【背景】真菌单细胞培养在研究细胞异质性及细胞生长特性等方面十分重要,因此需要建立简单便捷的方法对真菌单细胞进行培养与观察。【目的】基于微流控建立一种真菌单细胞的捕获及培养方法,同时直观地对单细胞进行定位和实时观察。【方法】利用L-edit设计芯片图案并利用等离子键合的方法制备微流控芯片;通过注射泵将红酵母菌溶液及里氏木霉孢子溶液进样以实现单细胞捕获;采用台盼蓝染色法测定酵母细胞的存活率;利用显微镜对酵母单细胞及木霉孢子的萌发、生长、繁殖过程进行观察。【结果】所制备的芯片形状完好,可实现酵母或孢子的单细胞捕获;酵母的捕获率为25.00%±1.38%;分别于0、2、4、6h对酵母进行观察,可看到酵母出芽过程;培养至48h,芯片上酵母细胞的存活率与游离培养条件下的存活率无显著性差异;分别于0、3、6、9 h对单个孢子进行观察,可以看到孢子萌发以及菌丝生长情况,且直至120h菌丝仍在生长。【结论】设计并制备了一种用于真菌单细胞捕获及定位培养的微流控芯片,这是此种芯片在真菌单细胞培养中的首次应用。细胞可在此微流控芯片上正常生长至少2 d,并可实现5 d及更长时间的培养,此方法可对真菌单细胞进行直观、定位的实时观察,有望用于多种微生物单细胞的生理、遗传性状研究,以及原生质体融合育种研究。     

Imaging of cell membraneous and cytoskeletal structures with a scanning tunneling microscope

The first observation of unstained cell membraneous structures by a scanning tunneling microscope is reported. An adhesive preparation method was used for imaging human medulloblastoma cells from the cell line TE 671 and oocytes from the clawed toad Xenopus laevis. The images show filaments, stacks of molecules and hilly structures. The possible identify of the filamentous structures is discussed, although the observed structures cannot yet be fully characterized. The work suggests possible future experiments on various biological structures in their natural environment.     

Regulation of Mitosis in Living Cells. I. Mitosis under Controlled Conditions

A quantitative method has been devised to study mitosis in vitro by phase contrast and polarization microscopy. Mitosis in cell-wall-free endosperm cells of Haemanthus kathrinar Baker (the African blood lily) has been divided into 18 arbitrary stages or events. The time course for the various stapes, as well as the percentage of cells that proceed from one stage to another during a four hour observation period, are presented. Cells that were in prophase when selected for study proceeded from nuclear membrane breakdown to melaphase in 60 minutes and remained in melaphase for 30 minutes. Only 13 minutes was required to proceed from onset of anaphase to mid-anaphasc. Mid-anaphase provides a clear and precise baseline for determining the time required for succeeding stages to appear. The cell plate made its appearance 40 minutes after mid-anaphase and was completely formed 20 minutes later. The nuclear membranes also became evident at this latter time and nucleoli were visible 30 minutes later. Thus, the average time for a cell observed initially in prophase to proceed from nuclear membrane breakdown to formation of two daughter cells was just over three hours. A high percentage of cells that were in late prophase or later stages of mitosis at the time of initial observation completed mitosis during the observation period. The effect of the length of time a cell is subjected to experimental conditions upon its subsequent behaviour is assessed. These results form the basis for future studies of the effects of chemicals, particularly herbicides, upon cells in mitosis as observed in vitro by phase contrast and polarization microscopy.     

Detection of local ATP release from activated platelets using cell surface-attached firefly luciferase

We have developed a method for measuring the local concentrationof ATP at the extracellular surface of live cells. This method relieson the specific attachment to the cell surface of a chimeric proteinthat consists of the IgG-binding domain ofStaphylococcus aureus protein A fusedin-frame with the complete sequence for firefly luciferase (proA-luc).Expression of proA-luc in Escherichia coli and its one-step affinity purification arestraightforward. Attachment to cells is demonstrated to be specific andantibody dependent using several suspended and adherent cell types.Light production by cell surface-attached luciferase is continuous, linearly related to ATP concentration, and sufficient to provide nanomolar sensitivity. The spatial resolution of this method enables the observation of strictly local changes in extracellular ATP duringits secretion from activated platelets. Furthermore, the activity ofcell-attached luciferase is relatively refractory to the inclusion ofnucleotidases in the medium, arguing for its effectiveness in cellsystems possessing potent ecto-ATPases.

     

The Role of Cell Wall Revealed by the Visualization of Saccharomyces cerevisiae Transformation

Transformation is an indispensable method for the manipulation of Saccharomyces cerevisiae cell. The spf1 cell, in which the gene encoding an endoplasmic reticulum-located P-type ATPase is deleted, has been known to show the high-transformation phenotype. In this study, fluorescent microscopic observation of transformation process of S. cerevisiae using plasmid DNA labelled with fluorescent DNA probe, YOYO-1, suggested that the spf1 cell absorbed more plasmid DNA on cellular surface than did the wild-type cell and the unwashed cell did more plasmid DNA than the washed cell. The amounts of the absorbed DNA correlated with the transformation efficiency (number of transformants per μg plasmid DNA) and frequency (transformation efficiency per viable cell number). The high-transformation phenotype of spf1 cell and the effect of heat shock, which effectively induces the transformation of intact cell, disappeared upon cell wall digestion. Electron microscopic observation of the transformation process using negatively charged Nanogold as a mimic of plasmid DNA supported the result obtained using YOYO-1 and implied that plasmid DNA enters into cell together with membrane structure. These data strongly suggest that during the transformation of intact cell, plasmid DNA is initially absorbed on the cell wall, passes through the cell wall with the aid of heat shock, reaches to the membrane, and enters into the cell together with the membrane structure and that the capacity of the cell wall to absorb DNA is at least one of the determinants of transformation efficiency and frequency.     

Approximate Confidence Interval on Ratio of Two Variances in a Two-way Crossed Model

The model considered is a two-factor cross-classification variance components model with one observation per cell. Let the two factors be A and B, the problem is to obtain an approximate confidence interval for the ratio of variance component A over variance component B. In this paper, a method of solving this problem is established and simulations are performed to check the method.     

Long term observation of gel-entrapped microbial cells in a microscope reactor

Summary A method is described for the on-line observation of immobilized, growing microorganisms in a microreactor mounted on a light microscope to determine several physiological parameters such as cell size and colony size, growth rates, spatial and temporal distribution of cells which are entrapped within transparent gels. The results can be used for modelling growth and diffusion behaviour in biocatalysts to optimize environmental and industrial applications of immobilized cells.     

A Bacterial Cell Wall Stain1

A new method is given to stain bacterial cell walls, especially of Escherichia coli and Bacillus cereus. The cells are smeared in water on a slide and, as soon as air-dry, are stained 3-4 minutes with a 1 % aqueous solution of new fuchsin. The smear is washed with water until the stain ceases to run and is then allowed to air dry. The slide is placed on a 50°C. warm plate for 10-20 seconds, and the smear is then covered with a thin film of a 1-2% solution of Congo red at a pH of about 9.5. The smear is ready for observation as soon as dry or it may be washed with water if desired before observation.