Methods in cell science : an official journal of the Society for In Vitro Biology最新文献_第4页

A rapid and reliable flow cytometric method for determining Hsp70 levels in tobacco protoplasts. 测定烟草原生质体中 Hsp70 水平的快速可靠的流式细胞计数法。

Methods in cell science : an official journal of the Society for In Vitro Biology Pub Date : 2003-01-01 DOI: 10.1007/s11022-004-2878-z

Marianne J Cronjé, Marisha Snyman, Liza Bornman, Iona E Weir

{"title":"A rapid and reliable flow cytometric method for determining Hsp70 levels in tobacco protoplasts.","authors":"Marianne J Cronjé, Marisha Snyman, Liza Bornman, Iona E Weir","doi":"10.1007/s11022-004-2878-z","DOIUrl":"10.1007/s11022-004-2878-z","url":null,"abstract":"Current methods to determine heat shock protein (Hsp) synthesis or accumulation in plant cells, such as Western blotting and biometabolic labelling are either indirectly quantitative, labour-intensive or biohazardous. An optimal flow cytometric protocol was developed to measure the intracellular Hsp70/Hsc70 levels in tobacco protoplasts. After heat treatments, protoplasts were fixed in 2% paraformaldehyde-phosphate-buffered saline and dehydrated overnight in methyl cellusolve, followed by permeabilization with Triton X-100 (0.1% in Protoplast Wash Fluid). Immunolabelling of Hsp70/Hsc70 was done for 1 hour with a mouse monoclonal antibody and detected by R-Phycoerythrin-conjugated goat anti-mouse IgG using flow cytometry. Flow cytometry detected a significant 1.2-fold increase in Hsp70/Hsc70 accumulation (P < 0.001) in protoplasts, while Western blotting, quantified by image analysis, showed induction under similar conditions but at lower significance (P < 0.05). The coefficients of variance for flow cytometry and Western blotting were 30.7 and 49.8 respectively. Optimum temperature of heat-induced Hsp70/Hsc70 accumulation in tobacco protoplasts occurred at 40 degrees C. Flow cytometry is proposed as a quantitative, more reproducible and rapid alternative to Western blotting for the detection of Hsp70 accumulation in plant cells.","PeriodicalId":80082,"journal":{"name":"Methods in cell science : an official journal of the Society for In Vitro Biology","volume":"25 3-4","pages":"237-46"},"PeriodicalIF":0.0,"publicationDate":"2003-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1007/s11022-004-2878-z","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"25203832","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: 4

A new method to obtain epithelial and stromal explants from human Corneo-Scleral Discs for the routine culture of corneal epithelial and fibroblast cells. 一种从人角膜巩膜盘获得上皮和间质外植体的新方法，用于角膜上皮和成纤维细胞的常规培养。

Methods in cell science : an official journal of the Society for In Vitro Biology Pub Date : 2003-01-01 DOI: 10.1007/s11022-004-6793-0

Sim F Webb, Sheila Davies, Richard Evans-Gowing, George Duncan

{"title":"A new method to obtain epithelial and stromal explants from human Corneo-Scleral Discs for the routine culture of corneal epithelial and fibroblast cells.","authors":"Sim F Webb, Sheila Davies, Richard Evans-Gowing, George Duncan","doi":"10.1007/s11022-004-6793-0","DOIUrl":"https://doi.org/10.1007/s11022-004-6793-0","url":null,"abstract":"Acquisition of human corneal cells for culture is hindered not only by the scarcity of donor tissues but also by some of the standard enzymatic and mechanical isolation techniques. Good yields have been reported from full-thickness explant and sclero-limbal pieces. However, due to their greater proliferative capacity, fibroblasts will encroach and subsequently overwhelm epithelial cultures whichever technique is used. The novel approach presented here is to minimise this by removal of the whole stroma from the epithelial layers at the outset. This is achieved by selective sectioning with the Webb mini-microtome developed in the Norwich Eye Research Laboratory. The microtome can be sterilised by alcohol spraying or autoclaving and is small enough to use in the culture hood. A selective cut in the region of the Bowman's membrane results in the isolation of the epithelium from the stroma and thus exposed, the basal epithelial layers are released from contact inhibition to allow growth. The stroma is further cut to produce multiple sections for the culture of fibroblasts. Both pure epithelial and stromal fibroblast cultures have been successfully generated in serum-enriched medium as well as defined serum-free media with growth supplements, from the corneo-scleral discs of donors of all ages.","PeriodicalId":80082,"journal":{"name":"Methods in cell science : an official journal of the Society for In Vitro Biology","volume":"25 3-4","pages":"167-76"},"PeriodicalIF":0.0,"publicationDate":"2003-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1007/s11022-004-6793-0","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"25204576","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: 2

Comparing protein stabilities during zebrafish embryogenesis. 斑马鱼胚胎发生过程中蛋白质稳定性的比较。

Methods in cell science : an official journal of the Society for In Vitro Biology Pub Date : 2003-01-01 DOI: 10.1023/B:MICS.0000006895.03556.9b

Thomas Becker, Michael Bossenz, Baris Tursun, Anne Schlüter, Marvin A Peters, Catherina G Becker, Heather P Ostendorff, Ingolf Bach

引用次数: 4

Coulter counter use in the enumeration of muscle and fat stem cells. 库尔特计数器在肌肉和脂肪干细胞计数中的应用。

Methods in cell science : an official journal of the Society for In Vitro Biology Pub Date : 2003-01-01 DOI: 10.1007/978-3-642-45207-9_19

M. Fernyhough, D. Helterline, J. Vierck, R. Hill, M. Dodson

引用次数: 6

Analysis of intracellular cytokines using flowcytometry. 流式细胞术分析细胞内细胞因子。

Methods in cell science : an official journal of the Society for In Vitro Biology Pub Date : 2002-01-01 DOI: 10.1023/a:1024177428018

Sunil K Arora

引用次数: 0

Nitric oxide mediated modulation of free radical generation response in the rat polymorphonuclear leukocytes: a flowcytometric study. 一氧化氮介导的大鼠多形核白细胞自由基生成反应的调节:一项流式细胞术研究。

Methods in cell science : an official journal of the Society for In Vitro Biology Pub Date : 2002-01-01 DOI: 10.1023/a:1024197915723

Madhu Dikshit, Prashant Sharma

{"title":"Nitric oxide mediated modulation of free radical generation response in the rat polymorphonuclear leukocytes: a flowcytometric study.","authors":"Madhu Dikshit, Prashant Sharma","doi":"10.1023/a:1024197915723","DOIUrl":"https://doi.org/10.1023/a:1024197915723","url":null,"abstract":"Nitric oxide (NO) synthesis and free radical generation from polymorphonuclear leukocytes (PMNs) play an important role in several pathological conditions. It is therefore important to understand the regulatory mechanisms of free radical generation from PMNs. Flowcytometry can be used to assess generation of reactive oxygen and nitrogen species from PMNs by using fluorescent probes. In the present study regulation of NO synthesis in the control and lipopolysaccharide (LPS) treated rat PMNs has been investigated. Free radical generation was assessed by flow cytometry using a dye, 2'7'-dichlorodihydrofluorescein diacetate (DCFDA), dihydrorhodamine-123 (DHR) and 4,5-diaminofluorescein diacetate (DAF). Superoxide dismutase (SOD), and catalase significantly attenuated the arachidonic acid (AA, 1 x 10(-6) M) induced free radical generation, while 4-aminobenzoicacid hydrazide (ABH), myeloperoxidase (MPO) inhibitor had no significant effect. Intracellular and extracellular calcium levels also modulated FR generation. AA induced free radical generation from PMNs was also enhanced significantly after LPS treatment. NO synthase (NOS) inhibitors, aminoguanidine (AG) and 7-nitroindazole (NI) inhibited arachidonic acid induced free radical generation from LPS treated PMNs, while in control PMNs NOS inhibition had no effect. Augmentation of free radical generation from rat PMNs following LPS treatment seems to be regulated by NO.","PeriodicalId":80082,"journal":{"name":"Methods in cell science : an official journal of the Society for In Vitro Biology","volume":"24 1-3","pages":"69-76"},"PeriodicalIF":0.0,"publicationDate":"2002-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1023/a:1024197915723","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"22444206","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: 0

Membrane oxidative damage and apoptosis in cervical carcinoma cells of patients after radiation therapy. 宫颈癌放疗后细胞膜氧化损伤及细胞凋亡的研究。

Methods in cell science : an official journal of the Society for In Vitro Biology Pub Date : 2002-01-01 DOI: 10.1023/A:1024145931652

S. Bhosle, B. Pandey, N. Huilgol, K. Mishra

引用次数: 17

Flowcytometric detection of PNH defect and response to therapy in aplastic anemia patients. 再生障碍性贫血患者PNH缺陷的流式细胞检测及对治疗的反应。

Methods in cell science : an official journal of the Society for In Vitro Biology Pub Date : 2002-01-01 DOI: 10.1007/978-94-017-0623-0_12

N. Varma, S. Varma, H. Vohra, K. Malik, G. Garewal

引用次数: 3

Cell array coupled with laser scanning cytometry allows easy analysis of changes in cyclin expression during the cell cycle. An application of cell array system. 细胞阵列与激光扫描细胞术相结合，可以轻松分析细胞周期蛋白表达的变化。小区阵列系统的一个应用。

Methods in cell science : an official journal of the Society for In Vitro Biology Pub Date : 2002-01-01 DOI: 10.1007/978-94-017-0623-0_6

T. Furuya, Morihito Takita, S. Tsunoda, S. Kawauchi, T. Hirano, A. Oga, K. Sasaki

引用次数: 1

A flow cytometric approach to assess phytoplankton respiration. 流式细胞术评估浮游植物呼吸作用。

Methods in cell science : an official journal of the Society for In Vitro Biology Pub Date : 2002-01-01 DOI: 10.1023/a:1024110418448

Gérald Grégori, Michel Denis, Dominique Lefèvre, Beatriz Beker

{"title":"A flow cytometric approach to assess phytoplankton respiration.","authors":"Gérald Grégori, Michel Denis, Dominique Lefèvre, Beatriz Beker","doi":"10.1023/a:1024110418448","DOIUrl":"https://doi.org/10.1023/a:1024110418448","url":null,"abstract":"Microbial respiration in the ocean is considered as the major process representative of the organic matter biological oxidation. The corresponding metabolic CO2 production was estimated to be about 22 Pg C y(-1). However, the in situ respiration rate is generally too low (by several orders of magnitude) to be accessible to the available direct measurement methods. Some fluorescent probes, such as DiOC6(3) (Molecular Probes, USA) have been shown to be very sensitive to changes in the proton electrochemical potential difference (DeltamuH+), characterising mitochondrial and plasmic membranes bearing the cell respiratory system in eukaryotic and prokaryotic cells respectively. In mitochondria, DeltamuH+ is linked to the flux of oxygen uptake by a linear relationship. To our knowledge, no such relationship has been established in the case of whole marine cells. In the present work, we addressed the dark respiration rate of the Chlorophyceae Dunaliella tertiolecta (Butcher) in axenic cultures, both directly by using a highly sensitive oxygraph (Oroboros) and by staining cells with DiOC6(3). We found and standardized a linear relationship between oxygen uptake by D. tertiolecta and its green fluorescence induced by DiOC6(3), enabling the determination by flow cytometry of the respiration rate of D. tertiolecta.","PeriodicalId":80082,"journal":{"name":"Methods in cell science : an official journal of the Society for In Vitro Biology","volume":"24 1-3","pages":"99-106"},"PeriodicalIF":0.0,"publicationDate":"2002-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1023/a:1024110418448","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"22444714","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