Methods in cell science : an official journal of the Society for In Vitro Biology最新文献_第2页

Analysis of cytosolic and lysosomal pH in apoptotic cells by flow cytometry. 流式细胞术分析凋亡细胞胞浆及溶酶体pH值。

Methods in cell science : an official journal of the Society for In Vitro Biology Pub Date : 2003-01-01 DOI: 10.1007/s11022-004-8228-3

Cathrine Nilsson, Katarina Kågedal, Uno Johansson, Karin Ollinger

{"title":"Analysis of cytosolic and lysosomal pH in apoptotic cells by flow cytometry.","authors":"Cathrine Nilsson, Katarina Kågedal, Uno Johansson, Karin Ollinger","doi":"10.1007/s11022-004-8228-3","DOIUrl":"https://doi.org/10.1007/s11022-004-8228-3","url":null,"abstract":"Several reports indicate that the cytosol is acidified during apoptosis although the mechanism is not yet fully elucidated. The most acidic organelle found in the cell is the lysosome, raising the possibility that lysosomal proton release may contribute to the cytosolic acidification. We here describe methods for measurement of the cytosolic and lysosomal pH in U937 cells by a dual-emission ratiometric technique suitable for flow cytometry. Cytosolic pH was analysed in cells loaded with the fluorescent probe BCECF, while lysosomal pH was determined after endocytosis of FITC-dextran. Standard curves were obtained by incubating cells in buffers with different pH in the presence of the proton ionophore nigericin. Apoptosis was induced by exposure of cells to 10 ng/ml TNF-alpha for 4 h, and apoptotic cells were identified using a fluorescent marker for active caspases. By gating of control and apoptotic cells, the cytosolic and lysosomal pH were calculated in each population. The cytosolic pH was found to decrease from 7.2+/-0.1 to 5.8+/-0.1 and the lysosomal increased from 4.3+/-0.4 to 5.2+/-0.3. These methods will be useful in future attempts to evaluate the involvement of lysosomes in the acidification of the cytosol during apoptosis.","PeriodicalId":80082,"journal":{"name":"Methods in cell science : an official journal of the Society for In Vitro Biology","volume":"25 3-4","pages":"185-94"},"PeriodicalIF":0.0,"publicationDate":"2003-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1007/s11022-004-8228-3","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"25203826","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: 129

Measurement of hemoglobin in long-term fixed erythroid cells: application development for cell science experiments in microgravity. 长期固定红细胞血红蛋白的测定:微重力条件下细胞科学实验的应用进展。

Methods in cell science : an official journal of the Society for In Vitro Biology Pub Date : 2003-01-01 DOI: 10.1007/s11022-004-2381-6

Kun Xu, Kerry L Davis, Arthur J Sytkowski

引用次数: 0

Sterile polystyrene culture dishes induce transformation of polyps into medusae in Aurelia aurita (Scyphozoa, Cnidaria). 无菌聚苯乙烯培养皿诱导水螅转化为水母(棘球纲，刺胞纲)。

Methods in cell science : an official journal of the Society for In Vitro Biology Pub Date : 2003-01-01 DOI: 10.1007/s11022-004-5592-y

Klaus Herrmann, Barbara Siefker, Stefan Berking

引用次数: 11

Epithelial cell cultures from Botryllus schlosseri palleal buds: accomplishments and challenges. 白葡萄孢嫩芽上皮细胞培养:成就与挑战。

Methods in cell science : an official journal of the Society for In Vitro Biology Pub Date : 2003-01-01 DOI: 10.1007/s11022-004-2087-9

Claudette Rabinowitz, Baruch Rinkevich

{"title":"Epithelial cell cultures from Botryllus schlosseri palleal buds: accomplishments and challenges.","authors":"Claudette Rabinowitz, Baruch Rinkevich","doi":"10.1007/s11022-004-2087-9","DOIUrl":"https://doi.org/10.1007/s11022-004-2087-9","url":null,"abstract":"This study focuses on recent improvement in epithelial monolayer cultures originating from whole extirpated Botryllus schlosseri (Urochordata) buds. Buds (n = 2,000) were taken at different ('A' to 'D') blastogenic stages. We tested the suitability of 35 combinations of various substrates and media on attachment, cell spread, epithelial growth frequencies and on monolayer lifespans. Under favorable conditions, cultured buds at blastogenic stages 'B' to 'D' (but not stage 'A') started to attach to the substrates following a 3-day transient period that leads to formation of spheres and attached monolayers. Substrate type is important for the attachment and the development of monolayers. Under various culture conditions, some of stages 'B' and 'C' buds develop (3-20 days) one or more large (1 mm diameter) spheres. Stage 'D' buds develop monolayers (up to 20% of buds) without going through a sphere phase. Neither spheres nor attached monolayers of epithelium were observed in stage 'A' bud cultures. Spheres grew at a rate of 60 microm in diameter per day using specific medium types and did not attach unless the appropriate substrate was present. When attached, epithelial monolayers expanded at a rate of 200 microm in diameter per day, for 3-15 days, and subsequently detached and died. Sixteen types of media were tested. Medium and substrate combinations were found to determine epithelial lifespan. These results revealed significant improvements in the culture of epithelial monolayers from Botryllus palleal buds. However, an early senescence of the developed epithelial sheets (up to two weeks from onset of appearance) may indicate an internal ageing clock that should be taken into consideration in future approaches.","PeriodicalId":80082,"journal":{"name":"Methods in cell science : an official journal of the Society for In Vitro Biology","volume":"25 3-4","pages":"137-48"},"PeriodicalIF":0.0,"publicationDate":"2003-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1007/s11022-004-2087-9","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"25204573","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: 22

Development and characterisation of pilchard (Sardinops sagax neopilchardus) cell lines derived from liver and heart tissues. 来源于肝脏和心脏组织的沙丁鱼(Sardinops sagax neopilchardus)细胞系的发育和特性。

Methods in cell science : an official journal of the Society for In Vitro Biology Pub Date : 2003-01-01 DOI: 10.1007/s11022-004-9801-5

Lynette M Williams, Mark St J Crane, Nicholas Gudkovs

{"title":"Development and characterisation of pilchard (Sardinops sagax neopilchardus) cell lines derived from liver and heart tissues.","authors":"Lynette M Williams, Mark St J Crane, Nicholas Gudkovs","doi":"10.1007/s11022-004-9801-5","DOIUrl":"https://doi.org/10.1007/s11022-004-9801-5","url":null,"abstract":"Two cell lines have been established from juvenile pilchards (Sardinops sagax neopilchardus) caught in waters off the Victorian coast of Australia. Following establishment of primary cultures derived from different pilchard tissues, using various cell culture media, a pilchard liver (PL) cell line and a pilchard heart (PH) cell line have been maintained in Eagle's minimal essential medium supplemented with 10% foetal bovine serum for over four years. The cell lines have been cryopreserved in liquid nitrogen and can be recovered from storage with good cell viability. Stock cell cultures have been maintained at 20-22 degrees C on a continuous basis in normal atmosphere (100% air), with weekly subculture at a split ratio of 3:1. The origin of the cell cultures was confirmed by PCR analysis using primers designed to be specific for pilchard mitochondrial DNA. In addition, the liver cell line was cloned and both the parental cell line and clones thereof were shown to be susceptible to a broad range of marine and freshwater viral pathogens of fish.","PeriodicalId":80082,"journal":{"name":"Methods in cell science : an official journal of the Society for In Vitro Biology","volume":"25 3-4","pages":"105-13"},"PeriodicalIF":0.0,"publicationDate":"2003-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1007/s11022-004-9801-5","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"25032610","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: 11

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

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