Methods in cell science : an official journal of the Society for In Vitro Biology最新文献_第3页

Fluorescence microplate assay for the detection of oxidative burst products in tobacco cell suspensions using 2',7'-dichlorofluorescein. 使用 2',7'-二氯荧光素检测烟草细胞悬浮液中氧化猝灭产物的荧光微孔板测定法。

Methods in cell science : an official journal of the Society for In Vitro Biology Pub Date : 2003-01-01 DOI: 10.1007/s11022-004-3851-6

Isak B Gerber, Ian A Dubery

引用次数: 40

Isolation and primary culture of gill and digestive gland cells from the common mussel Mytilus edulis. 普通贻贝蚌鳃和消化腺细胞的分离与原代培养。

Methods in cell science : an official journal of the Society for In Vitro Biology Pub Date : 2003-01-01 DOI: 10.1007/s11022-004-8227-4

Jérôme Faucet, Manuelle Maurice, Béatrice Gagnaire, Tristan Renault, Thierry Burgeot

{"title":"Isolation and primary culture of gill and digestive gland cells from the common mussel Mytilus edulis.","authors":"Jérôme Faucet, Manuelle Maurice, Béatrice Gagnaire, Tristan Renault, Thierry Burgeot","doi":"10.1007/s11022-004-8227-4","DOIUrl":"https://doi.org/10.1007/s11022-004-8227-4","url":null,"abstract":"As the marine mussel Mytilus edulis is commonly used as a sentinel species, it would be useful to develop a primary culture of the target organs most often in contact with the marine environment. This study reports an improved method for dissociating the digestive gland and gills of M. edulis and considers the effect of mussel storage on cell viability and functionality before culture initiation. Viability and enzymatic activities such as those of esterase and peroxidase were monitored by flow cytometry, a sensitive, objective technique allowing large volumes of cells to be counted within a short time. A primary culture of digestive gland showed more than 75% viability after 72 h. Mussels were maintained in an aquarium containing clean, oxygenated seawater at 12 degrees C for two days before culture initiation, and dissociation was performed mechanically and chemically with Ca-Mg-free saline to obtain digestive gland cells. Application of non-specific esterase activity, using fluorescein diacetate (FDA test) coupled with flow cytometry, characterised the functionality of digestive gland and gill cells in culture.","PeriodicalId":80082,"journal":{"name":"Methods in cell science : an official journal of the Society for In Vitro Biology","volume":"25 3-4","pages":"177-84"},"PeriodicalIF":0.0,"publicationDate":"2003-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1007/s11022-004-8227-4","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"25204577","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 23

Immunocytochemistry as a tool for zebrafish developmental neurobiology. 免疫细胞化学在斑马鱼发育神经生物学研究中的应用。

Methods in cell science : an official journal of the Society for In Vitro Biology Pub Date : 2003-01-01 DOI: 10.1023/B:MICS.0000006894.43940.b1

Alicia E Novak, Angeles B Ribera

引用次数: 26

An in vitro method for analysis of chondrogenesis in limb mesenchyme from individual transgenic (hdf) embryos. 转基因(hdf)胚胎肢体间质软骨形成的体外分析方法。

Methods in cell science : an official journal of the Society for In Vitro Biology Pub Date : 2003-01-01 DOI: 10.1007/s11022-004-9803-3

Danielle M Gillotte, Patricia L Fox, Corey H Mjaatvedt, Stanley Hoffman, Anthony A Capehart

{"title":"An in vitro method for analysis of chondrogenesis in limb mesenchyme from individual transgenic (hdf) embryos.","authors":"Danielle M Gillotte, Patricia L Fox, Corey H Mjaatvedt, Stanley Hoffman, Anthony A Capehart","doi":"10.1007/s11022-004-9803-3","DOIUrl":"https://doi.org/10.1007/s11022-004-9803-3","url":null,"abstract":"The present study describes a simple, rapid protocol for culture for limb tissue from individual 10.5-day post coitum mouse embryos that supports cartilage differentiation over a six-day period. This technique differs from other commonly used methods utilizing pooled limb tissue in that: 1) forelimbs from individual embryos were used as donor tissue; 2) limb tissue was dissociated by very gentle enzymatic digest (0.1% trypsin, 5 min); and, 3) cell suspensions were plated at a lower density (1 x 10(7) vs. 2 x 10(7) cells/ml) in a reduced volume of 3-5 microl. Under these modified conditions to increase limb cell yield from each embryo, histochemical and immunohistochemical analyses demonstrated reproducible formation of precartilage aggregates and subsequent overt chondrogenesis over a predictable time course. Using this culture protocol, analysis of limb mesenchyme from heterozygous hdf embryos, which bear an insertional mutation of the Cspg2 gene encoding the core protein of the chondroitin sulfate proteoglycan, versican, revealed an overall similar chondrogenic potential to that observed for wild-type littermates. This technique readily enables in vitro culture of limb bud mesenchyme from individual mouse embryos at this developmental stage and may be utilized by investigators to study the effects of the hdf and other transgenic mutations on mammalian limb development in vitro.","PeriodicalId":80082,"journal":{"name":"Methods in cell science : an official journal of the Society for In Vitro Biology","volume":"25 3-4","pages":"97-104"},"PeriodicalIF":0.0,"publicationDate":"2003-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1007/s11022-004-9803-3","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"25032609","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 10

Establishment, growth, cryopreservation and species of origin identification of three cell lines from white sturgeon, Acipenser transmontanus. transmontanus白鲟三种细胞系的建立、生长、冷冻保存及种源鉴定。

Methods in cell science : an official journal of the Society for In Vitro Biology Pub Date : 2003-01-01 DOI: 10.1007/s11022-004-9120-x

Guitang Wang, Scott LaPatra, Lingbing Zeng, Zhengshan Zhao, Yuanan Lu

{"title":"Establishment, growth, cryopreservation and species of origin identification of three cell lines from white sturgeon, Acipenser transmontanus.","authors":"Guitang Wang, Scott LaPatra, Lingbing Zeng, Zhengshan Zhao, Yuanan Lu","doi":"10.1007/s11022-004-9120-x","DOIUrl":"https://doi.org/10.1007/s11022-004-9120-x","url":null,"abstract":"Three cell lines derived from fin (WSF), head soft tissue (WSHST) and body muscle (WSBM) were established from white sturgeon (Acipenser transmontanus). Characterization included determination of optimal growth kinetics, karyotyping, and mitochondrial ribosomal RNA (rRNA) genotyping. The primary cultures of these cells were generated by the explant technique using the L-15 medium supplemented with 20% fetal bovine serum and epidermal/fibroblast growth factors. The cells grew between 15-30 degrees C, but optimal growth occurred at 25 degrees C with a doubling time of 48 hours. The cell lines can be readily maintained in-vitro and have been subcultured over 35 times. Following cryopreservation in liquid nitrogen, thawed cells exhibit a viability of > 90% after a 16-month storage period. Chromosomal typing of these cell lines at their 17th passage revealed a chromosomal distribution of 242 to 278 with an apparent peak ranging from 250 to 260. Polymerase chain reaction amplification of mitochondrial 16S ribosomal RNA and sequence analysis indicated 100% identity of the sequences found in the cell lines with those found in the source animal, confirming that the cell lines were of A. transmontanus origin.","PeriodicalId":80082,"journal":{"name":"Methods in cell science : an official journal of the Society for In Vitro Biology","volume":"25 3-4","pages":"211-20"},"PeriodicalIF":0.0,"publicationDate":"2003-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1007/s11022-004-9120-x","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"25203829","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 43

Neuron-specific gene manipulations to transparent zebrafish embryos. 透明斑马鱼胚胎的神经元特异性基因操作。

Methods in cell science : an official journal of the Society for In Vitro Biology Pub Date : 2003-01-01 DOI: 10.1023/B:MICS.0000006850.81427.ed

Tomoyuki Yoshida, Masayoshi Mishina

{"title":"Neuron-specific gene manipulations to transparent zebrafish embryos.","authors":"Tomoyuki Yoshida, Masayoshi Mishina","doi":"10.1023/B:MICS.0000006850.81427.ed","DOIUrl":"https://doi.org/10.1023/B:MICS.0000006850.81427.ed","url":null,"abstract":"To investigate the molecular basis of neural network formation, we introduced a novel double-cassette vector approach for visualizing and manipulating neuronal development in living zebrafish embryos. Two genes are physically linked in the double-cassette vector system, which ensures co-expression of an effector-protein and an EGFP-reporter in the same neuron. By generating transgenic enhanced green fluorescent protein (EGFP) expressing zebrafish lines, we first established that EGFP under control of either the olfactory marker protein (OMP) gene promoter or the nicotinic acetylcholine receptor beta3 (nAChRbeta3) gene promoter, directed strong EGFP expression to the olfactory sensory neurons and the retinal ganglion cells (RGCs), respectively. These transgenic lines allowed the visualization of the development of the entire olfactory sensory neurons and RGCs in vivo. By injection of vectors with EGFP under control of either the OMP or the nAChRbeta3 gene promoter, we followed the development of individual olfactory sensory neurons and RGCs. The double-cassette expression vector strategy enabled us to clarify the roles of protein kinase A (PKA) and glycogen synthase kinase-3beta (GSK-3beta) in the development of olfactory sensory neurons and RGCs. The combination of visualization and neuron-specific gene manipulation provides a powerful reverse genetic in vivo approach for the study of genes of interest in neural differentiation, axonal pathfinding, and synaptogenesis.","PeriodicalId":80082,"journal":{"name":"Methods in cell science : an official journal of the Society for In Vitro Biology","volume":"25 1-2","pages":"15-23"},"PeriodicalIF":0.0,"publicationDate":"2003-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1023/B:MICS.0000006850.81427.ed","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"24177901","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 19

Practical procedures for ectopic induction of gene expression in zebrafish embryos using Bhc-diazo-caged mRNA. 利用bhc -重氮笼mRNA异位诱导斑马鱼胚胎基因表达的实用方法。

Methods in cell science : an official journal of the Society for In Vitro Biology Pub Date : 2003-01-01 DOI: 10.1023/B:MICS.0000006846.13226.38

Hideki Ando, Hitoshi Okamoto

引用次数: 22

Preparation of chromosomes from plant leaf meristems for karyotype analysis and in situ hybridization. 从植物叶片分生组织中提取染色体进行核型分析和原位杂交。

Methods in cell science : an official journal of the Society for In Vitro Biology Pub Date : 2003-01-01 DOI: 10.1007/s11022-004-5620-y

Kesara Anamthawat-Jónsson

{"title":"Preparation of chromosomes from plant leaf meristems for karyotype analysis and in situ hybridization.","authors":"Kesara Anamthawat-Jónsson","doi":"10.1007/s11022-004-5620-y","DOIUrl":"https://doi.org/10.1007/s11022-004-5620-y","url":null,"abstract":"A reliable method for preparing metaphase chromosomes from plant leaf tissues is described. The chromosomes are suitable for karyotype analysis and gene mapping by fluorescence in situ hybridisation (FISH). The method is based on enzymatic digestion of young leaf tissues (shoot-tips) after which the resulting protoplasts are treated hypotonically before being dropped onto microscopic slides. Compared to root-tip chromosomes, leaf chromosomes tend to be longer, or less condensed, and hence more karyotypically differentiated. Metaphase index in young leaf tissues is also very high. Metaphase spread consists of evenly and well-distributed chromosomes and this allows accurate counting. The plant used to demonstrate this method is birch (Betula L.), a group of tree species that has extremely small chromosomes. Root-tip chromosomes of these plants are difficult to obtain, as cutting does not produce roots readily. Seedling chromosomes do not represent the same genomic constitution as their mother trees due to introgressive hybridisation. Furthermore, sample collection in the field is convenient and actively growing leaf buds are available throughout the growing season. FISH experiments with these leaf chromosomes also give good results comparable to those obtained with root-tip chromosomes or even better as mapping on long or extended chromosomes has high resolution in general. Mapping of the 16S-28S ribosomal genes on birch leaf chromosomes has been shown to differentiate between birch species and therefore can accurately confirm their interspecific hybrids.","PeriodicalId":80082,"journal":{"name":"Methods in cell science : an official journal of the Society for In Vitro Biology","volume":"25 3-4","pages":"91-5"},"PeriodicalIF":0.0,"publicationDate":"2003-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1007/s11022-004-5620-y","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"25032606","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 51

Double labeling of neurons by retrograde axonal tracing and non-radioactive in situ hybridization in the CNS of adult zebrafish. 用逆行轴突示踪和非放射性原位杂交技术在成年斑马鱼中枢神经系统中对神经元进行双重标记。

Methods in cell science : an official journal of the Society for In Vitro Biology Pub Date : 2003-01-01 DOI: 10.1023/B:MICS.0000006848.57869.4c

Bettina C Lieberoth, Catherina G Becker, Thomas Becker

{"title":"Double labeling of neurons by retrograde axonal tracing and non-radioactive in situ hybridization in the CNS of adult zebrafish.","authors":"Bettina C Lieberoth, Catherina G Becker, Thomas Becker","doi":"10.1023/B:MICS.0000006848.57869.4c","DOIUrl":"https://doi.org/10.1023/B:MICS.0000006848.57869.4c","url":null,"abstract":"A number of genes affecting axonal projections are currently being identified in zebrafish mutant screens. Analyzing the expression of these genes in the adult brain in relation to specific neuronal populations could yield insights into new functional contexts, such as the successful axonal regeneration in adult zebrafish. Here, we provide a relatively simple procedure for non-radioactive in situ hybridization in sections of adult zebrafish brains in combination with retrograde axonal tracing using the fluorescent neuronal tracer rhodamine dextran amine (RDA). A lesion is inflicted on the spinal cord of adult zebrafish and a crystal of RDA is then applied to the lesion site resulting in retrograde labeling of neurons in the brain through their spinal axons. Six to eighteen days later fish are perfusion-fixed, and in situ hybridization is carried out on vibratome-cut floating sections using a protocol simplified from that used for whole-mounted zebrafish embryos. This procedure leads to robust double labeling of axotomized neurons with RDA and an in situ hybridization signal for the growth-associated protein 43 (GAP-43). This method can be used to identify gene expression in specific populations of projection neurons and to detect changes in gene expression in axotomized neurons in the CNS of adult zebrafish.","PeriodicalId":80082,"journal":{"name":"Methods in cell science : an official journal of the Society for In Vitro Biology","volume":"25 1-2","pages":"65-70"},"PeriodicalIF":0.0,"publicationDate":"2003-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1023/B:MICS.0000006848.57869.4c","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"24177336","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 18

Cobblestone area measuring (CAM) assay: a new way of assessing the potential of human haemopoietic stem cells. 鹅卵石面积测定法:一种评估人造血干细胞潜能的新方法。

Methods in cell science : an official journal of the Society for In Vitro Biology Pub Date : 2003-01-01 DOI: 10.1007/s11022-004-9123-7

Dimitrios G Bouzianas

{"title":"Cobblestone area measuring (CAM) assay: a new way of assessing the potential of human haemopoietic stem cells.","authors":"Dimitrios G Bouzianas","doi":"10.1007/s11022-004-9123-7","DOIUrl":"https://doi.org/10.1007/s11022-004-9123-7","url":null,"abstract":"A new in vitro way of scoring the potential (proliferative- differentiative) of human haemopoietic stem cells (HHSC) is presented. We have applied it in the investigation of HHSC behaviour in normal bone marrow specimens under culture conditions. This system describes a modification of some existing culture assays for primitive HHSC function, and proliferation of the cobblestone area forming cell (CAFC) was determined by counting the cobblestone areas (CA) produced every week. The main steps of the assay are: (a) culture of MNC fraction on pre-established fibroblastic confluent layers from the M2.10B4 mouse cell line; (b) weekly counting of CA over a period of 5 weeks; (c) weekly in situ observation of CA morphology; (d) enumeration of secondary progenitors per CA, in the 5th week. The CAM index, expressing the value of the CA kinetic line slope and the number of secondary progenitors per CA were evaluated as factors indicating HHSC proliferative and differentiative potential, respectively. This culture system has the potential to serve as an in vitro assay of human stem cell transplantation. It could be applied to evaluating the potential of HHSC contained in haemopoietic collections of diverse origin, only in combination with measurements of the starting number of CAFC.","PeriodicalId":80082,"journal":{"name":"Methods in cell science : an official journal of the Society for In Vitro Biology","volume":"25 3-4","pages":"201-10"},"PeriodicalIF":0.0,"publicationDate":"2003-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1007/s11022-004-9123-7","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"25203828","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 3