A New Technique Facilitating Studies of Scant Cell Specimens

A simple method is described by which multiple cytological and cytochemical studies can be done on a clinical sample that contains relatively few cells. The cells are concentrated by centrifugation. The cell pellet is fixed, frozen and embedded in plastic. Thin (2-μm) sections are cut from the plastic. Thus, each cell may appear in several sections and many slides can be made from a single specimen. The advantages of this method over cytospins and Millipore filter preparations of cell suspensions are optimal utilization of all cells, excellent morphological and immunological preservation and ease and reproducibility of this technique.     

A Plastic Embedding Technique for Analyzing Fluorescent Dextran-Amine Labelled Neuronal Profiles

A plastic embedding technique employing fluorescently labelled dextran-amines is described. After application of tracer to cut nerves and appropriate transport time, animals were fixed in paraformaldehyde. Subsequently their brains were dissected, heads and brains were dehydrated, embedded in methacrylate and sectioned serially on a rotary microtome. Plastic sections allow high resolution of single neuron profiles and complete serial reconstruction of un-distorted sections, including embryos with large amounts of yolk. In conjunction with whole mount analysis and double labelling, this technique can accurately reveal the spatial relationships of nerve components throughout development.     

干细胞共培养技术在医学研究中的应用

细胞共培养技术是20世纪70年代后期发展起来的将不同种类、不同来源的细胞在同一个体系中进行培养、增殖的技术,该技术的诞生至今已经历了三十多年的时间,在共培养的细胞种类、共培养条件、共培养方法等方面均取得了很大的进展。其中,将骨髓间充质干细胞与其他种类细胞共培养,以诱导骨髓间充质干细胞定向分化的研究最为常见。该文对在神经、骨关节、心血管等系统疾病的替代治疗中有重要价值的共培养研究—骨髓间充质干细胞与不同种类的体细胞共培养作了重点介绍,以期为今后的工作提供借鉴和帮助。     

Orientation of Small Specimens for Cryosectioning

A simple technique is introduced to achieve symmetrically oriented frozen sections of small specimens such as young fish or frog larvae. Small samples are especially difficult to orient if they are already frozen to the chuck in a freezing microtome. Orientation of the sample in a mold filled with embedding medium prior to freezing permits sectioning as well as easy labeling and storage of the specimens. The use of a stereo microscope during orientation is optional.     

促细胞黏附生长的自组装肽水凝胶

随着细胞与组织工程的迅猛发展,能够促进细胞黏附、生长和分化的生物材料基质支架的研究日益重要。具有生物相容性且含水量超过99％的自组装肽水凝胶因其很好地符合理想的生物材料基质支架标准而备受重视。这类自我互补的两亲寡肽含50％的带电残基,并且以交替的离子亲水性和不带电的氨基酸残基周期性重复为特征;在其寡肽的氨基末端可用直接固相合成法修饰几个短序列生物活性模体进行功能化,用以促进不同细胞的黏附生长和靶向定位。现对自组装肽水凝胶的结构特征、自组装机制、对细胞黏附生长的影响以及未来自组装肽生物材料设计的目标进行综述．     

In Situ Embedding of Cell Monolayers Cultured on Plastic Surfaces for Electron Microscopy

Various procedures suitable for routine in situ embedding of cell monolayers were tested including: (1) the use of different Epon substitutes, (2) the use of different types of plas-ticware obtained from different sources, and (3) different methods of preparing capsules for sectioning. Different resins reacted differently with different plastics and type of preparation. Merck Epon substitute bound to most of the plastics tested. Ladd Epon substitute released cleanly from all plastics tested when a suitable method of preparation was used. The results show that for routine embedding of cell monolayers it is necessary to select an appropriate Epon substitute and method of preparation of capsules for the type of plasticware used. A routine method is described, with various alternative steps which can be applied when particular difficulties are encountered.     

A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning of such signaling can lead to many disorders. To understand cell-to-cell signaling, it is essential to study the spatial and temporal nature of the secreted molecules from the cell without disturbing the local environment. Various assays have been developed to study protein secretion, however, these methods are typically based on fluorescent probes which disrupt the relevant signaling pathways. To overcome this limitation, a label-free technique is required.In this paper, we describe the fabrication and application of a label-free localized surface plasmon resonance imaging (LSPRi) technology capable of detecting protein secretions from a single cell. The plasmonic nanostructures are lithographically patterned onto a standard glass coverslip and can be excited using visible light on commercially available light microscopes. Only a small fraction of the coverslip is covered by the nanostructures and hence this technique is well suited for combining common techniques such as fluorescence and bright-field imaging.A multidisciplinary approach is used in this protocol which incorporates sensor nanofabrication and subsequent biofunctionalization, binding kinetics characterization of ligand and analyte, the integration of the chip and live cells, and the analysis of the measured signal. As a whole, this technology enables a general label-free approach towards mapping cellular secretions and correlating them with the responses of nearby cells.     

新型细胞标记技术在酿酒酵母原生质体融合育种中的应用研究

本研究用酿酒酵母原生质体融合技术使两株酵母发生融合,通过用营养要求测定,交配型测定、细胞体积测定、DNA含量测定,筛选出13株融合株,再经免疫测定,得到同样的鉴定结果.表明免疫测定技术在酿酒酵母原生质体融合育种中作为亲本细胞标记是可行的,具有较高实用价值.     

A Double-Embedding Technique for Thin Tissue Membranes

A new double-embedding technique for thin tissue membranes is presented. This technique is useful for thin membranes such as mesenteric membranes from rodents, which usually measure only 10 μm in thickness. Several membranes are fixed and mounted on four needles located at the bottom of a plastic box. The box is filled with agarose at 50 C and then allowed to solidify. The agarose block is then removed, dehydrated in alcohol, cleared with Histo Petrol (isoparaffin hydrocarbons), permeated with paraffin and sectioned. The morphology is comparable to that obtained with methacrylate plastic embedding but is less time-consuming, less hazardous since no plastic hardener and activator are used and makes immunohistochemical studies easier.     

New Combination of Antibiotics for Treatment of Contaminated Specimens for Viral Studies

A study of a new combination of antibiotics (sodium oxacillin, colistin sulfate, and amphotericin B) has shown that 30 μg/ml of each in tissue culture maintenance medium can inhibit a group of the commonest bacterial and fungal contaminants while causing no observable cytopathology in rhesus monkey kidney, HeLa, or human amnion tissue cells. In addition, there was no inhibition of the titers of poliovirus, echovirus, coxsackievirus, and adenovirus in the three cell lines. Ten duplicate fecal samples, which remained contaminated with fungal or Pseudomonas organisms after treatment with penicillin and streptomycin, were treated with the new antibiotic combination. Contaminants were controlled in each, and a variety of viruses was recovered.     

微滴培养技术的新应用

微滴培养技术在卵母细胞培养和胚胎早期发育研究中有广泛的应用.近期研究发现这项技术又有新的应用范围.有科学家发现将该技术应用于胚胎干细胞可以实现高效传代,在精原干细胞培养和精子发生相关过程的研究方面也可取得很好效果,同时,对早期人类胚胎干细胞的分离过程的监控和对精原干细胞生长过程的观察也相对容易.这些研究结果显示,微滴培养技术在这些新领域的研究与常规培养方法相比具有独特的优势.回顾微滴培养技术的主要发展过程,重点探讨微滴培养技术的最新应用及其优点.     

动物细胞全能性的研究

在植物细胞全能性研究的基础上引出动物细胞全能性这一热点研究课题.介绍了动物细胞全能性的表现,分析了细胞全能性表现程度差异的原因,最后对动物细胞全能性的广泛应用及存在的问题进行探讨,并对细胞命运及其调控进行了展望.     

细胞融合技术的发展及应用

细胞工程是四大生物工程之一,细胞融合技术作为细胞工程的一项核心基础技术已在农业、医药、环保等领域取得了开创性的研究成果,而且应用领域不断扩大。细胞融合技术的不断改进一方面表现在融合剂上,另一方面体现在新方法上,再者体现在融合对象的不断扩展上。现在新的细胞融合方法正在尝试将各种物理、化学手段综合应用,使细胞融合的方法和手段向操作更为简便,便于量化研究,同时又能使融合率得到不断提高的方向发展。本文以细胞融合技术的发展历史为主线,对上述内容做了简要综述。     

A Soap Technique for Cell Separation to Study the Seed Coat of Sesbania punicea

A technique is described for separating plant cells used for morphological studies. The plant material is placed in a concentrated solution of olive oil castile soap for 1-2 days or more. The material is then thoroughly washed and placed between two glass slides. The upper glass slide is lifted from the lower one, then gently pressed down several times. Through this procedure Malpighian cells of the seed coat of Sesbania punicea. mesophyll cells of Euphorbia peplus and of Trifolium pratense and cortical cells of the aerial roots of Monstera deliciosa have been separated. Various shapes of the Malpighian cells of the Sesbania punicea seed coat can be observed along with intermediates.     

黄山药细胞培养初步研究

以黄山药叶片为外植体诱导愈伤组织,经多次继代培养筛选后进行细胞悬浮培养,研究了两种培养基对细胞生长动态的影响以及4种培养参数对细胞生长量的影响。结果表明,培养在含6-BA1 mg/L和2,4-D1 mg/L的MS液体培养基中的细胞生长效果较好,培养20 d左右可达最高生长点。较适宜的培养条件为:接种量0.8~1.0 g/100 mL;培养温度24℃~27℃;pH 5.2~5.8;蔗糖用量20~40 g/L;继代培养周期15~20 d。     

Development of New Cell Lines for Animal Cell Biotechnology

Abstract

Mammalian cell culture has been an important technique in laboratory-scale experimentation for many decades. Developments in large-scale culture have been due to the need to grow large numbers of cells to support the growth of viruses for vaccine production, and more recently, for growing hybridoma cells as a source of monoclonal antibody. Increasingly, however, pharmaceutical products such as hormones, enzymes, growth factors, and clotting factors are being produced from cell lines which have been manipulated by recombinant DNA techniques. It is clear, therefore, that the high cost of growing mammalian cells on a large scale does not necessarily prohibit their use for biotechnology, and indeed there is considerable evidence to suggest that animal cell biotechnology will continue to be a major growth area in the future.     

拉曼光谱分析技术在细胞生物学研究中的应用进展

细胞是生物体结构和功能的基本单位,自被发现以来新的研究方法不断涌现。单细胞拉曼光谱能提供细胞内核酸、蛋白质、脂质含量等大量信息,可在不损伤细胞的条件下实时动态地监测细胞分子结构变化,亦可获得细胞的“分子指纹”,具有敏感性高、实时检测、活样品不需固定或染色、不损伤细胞等众多特点。近年来国内外研究者将拉曼光谱应用于细胞药物处理、细胞水平疾病诊断、单细胞生命活动监测、亚细胞结构等研究,取得了不同程度的进展。随着研究的深入,拉曼光谱分析技术必将在干细胞,癌症研究、细胞分选、药物筛选等领域大有作为。     

荧光相关谱测量技术研究

荧光相关谱(fluorescence correlation spectroscopy,FCS)是对处于热平衡态条件下的荧光分子发出的荧光强度涨落进行时间相关处理的一种单分子检测方法,能够直接测量分子在溶液里的扩散系数和浓度.影响FCS测量扩散系数准确性的因素有分子量子效率,测量时间,样本折射率和温度偏差等.用FCS分别测量溶有荧光染料罗丹明6G(rhodamine 6G,Rh.6G)和青色素Cy5甘油水溶液的粘滞系数,实验结果表明:荧光分子的量子效率是影响测量准确性的重要因素,要求其每秒发射的光子数目(photon counts per second,cps)至少达到1 000(photons/s).     

Live Cell Multicolor Imaging of Lipid Droplets with a New Dye, LD540

A lipophilic dye based on the Bodipy fluorophore, LD540, was developed for microscopic imaging of lipid droplets. In contrast to previous lipid droplet dyes, it can spectrally be resolved from both green and red fluorophores allowing multicolor imaging in both fixed and living cells. Its improved specificity, brightness and photostability support live cell imaging, which was used to demonstrate by two-color imaging lipid droplet motility along microtubules.