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

{"title":"Cartilage extracellular matrix metabolism differs in serum and synovial fluid.","authors":"James A Martin,&nbsp;Andrew L Wilkey,&nbsp;Richard A Brand","doi":"10.1023/a:1024468101796","DOIUrl":"https://doi.org/10.1023/a:1024468101796","url":null,"abstract":"<p><p>Most cartilage explant culture studies assume conventional serum-supplemented growth media are biologically equivalent to the natural synovial fluid which baths cartilage in vivo. Few studies have systematically compared the effects of serum versus synovial fluid in culture. To address this assumption we conducted a series of studies to determine if cartilage matrix synthesis is significantly different in serum-based versus synovial fluid-based media. Normal bovine cartilage explants were cultured in DMEM either alone or supplemented with bovine serum or bovine synovial fluid. Matrix synthesis was measured with radiolabeling techniques. We then compared responses to insulin-like growth factor I (IGF-I, a stimulator of matrix synthesis), and interleukin-1beta (IL-1beta, an inhibitor of matrix synthesis). We observed significantly lower matrix synthesis activity in synovial fluid versus serum. Caution shoud be used in extrapolating studies of cartilage grown in media supplemented with serum rather than synovial fluid.</p>","PeriodicalId":80082,"journal":{"name":"Methods in cell science : an official journal of the Society for In Vitro Biology","volume":"24 4","pages":"139-43"},"PeriodicalIF":0.0,"publicationDate":"2002-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1023/a:1024468101796","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"22468973","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}

{"title":"Flow cytometry and its application in small animal oncology.","authors":"Katja Culmsee,&nbsp;Ingo Nolte","doi":"10.1023/a:1024133628927","DOIUrl":"https://doi.org/10.1023/a:1024133628927","url":null,"abstract":"<p><p>Flow cytometry measures multiple characteristics of single cells. The use of flow cytometry in the veterinary clinical laboratory has increased considerably during the past decade. The most common applications of flow cytometry in small animal oncology are measurement of DNA content in tumours and immunophenotyping of haematopoietic malignancies. DNA ploidy and S-phase rate of various tumours in dogs have been found to be independent predictor of patient outcome. In dogs with lymphomas immunophenotyping is recommended as a part of the patient work-up. Flow cytometry has shown to be a suitable method for immunophenotyping of canine lymphomas. However, it has not become a routine technique in small animal oncology yet. This report reviews the applications of flow cytometry in small animal oncology. Besides basic principles and technical aspects, the clinical relevance of DNA-analysis and immunophenotyping are discussed.</p>","PeriodicalId":80082,"journal":{"name":"Methods in cell science : an official journal of the Society for In Vitro Biology","volume":"24 1-3","pages":"49-54"},"PeriodicalIF":0.0,"publicationDate":"2002-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1023/a:1024133628927","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"22444202","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}

{"title":"Flow cytometry, an overview.","authors":"L Scott Cram","doi":"10.1023/a:1024198904819","DOIUrl":"https://doi.org/10.1023/a:1024198904819","url":null,"abstract":"","PeriodicalId":80082,"journal":{"name":"Methods in cell science : an official journal of the Society for In Vitro Biology","volume":"24 1-3","pages":"1-9"},"PeriodicalIF":0.0,"publicationDate":"2002-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1023/a:1024198904819","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"22444289","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}

{"title":"A method to isolate morphologically distinct clones of smooth muscle cells from human saphenous vein.","authors":"Zhongbiao Wang, Pulipaka J Rao, Manuel R Castresana, Walter H Newman","doi":"10.1023/a:1024461501028","DOIUrl":"10.1023/a:1024461501028","url":null,"abstract":"<p><p>The monoclonal theory of atherosclerosis postulates that a certain subpopulation of vascular smooth muscle cells (VSMC) is selectively expanded in response to pathological stimuli thereby contributing to the formation of atherosclerotic plaques. VSMC cloning experiments will be important in characterizing the phenotypic composition of VSMC in atherosclerotic plaques. However, the difficulty in cloning human VSMC is well recognized. Here a technique is described that produced multiple clones from human saphenous vein. The clones could be divided into two categories based on their distinctly different morphology: (1) spindle-shaped; and, (2) epithelioid-shaped. Each clone expressed smooth muscle-a-actin and calponin, two smooth muscle-specific differentiation markers. The clonal study presented here reports for the first time that phenotypically heterogeneous smooth muscle cells coexist within human saphenous veins.</p>","PeriodicalId":80082,"journal":{"name":"Methods in cell science : an official journal of the Society for In Vitro Biology","volume":"24 4","pages":"131-7"},"PeriodicalIF":0.0,"publicationDate":"2002-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1023/a:1024461501028","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"22468972","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}

{"title":"Purification of DNA for the transfection of a Spodoptera frugiperda cell line.","authors":"Jeffrey M Slack,&nbsp;Susan D Lawrence","doi":"10.1023/a:1024413604663","DOIUrl":"https://doi.org/10.1023/a:1024413604663","url":null,"abstract":"<p><p>Spodoptera frugiperda (Sf-9) cells have been widely used in baculovirus expression systems, transient gene expression studies and transgenic cell lines. These applications commonly require the transfection of bacterial plasmid DNA. One of the most reliable methods of preparing transfection-quality plasmid DNA is cesium chloride (CsCl) density gradient centrifugation. However, the traditional CsCl DNA purification is a long and laborious process. We have made a series of modifications to the traditional method that makes it faster, safer and easier. In the current study we demonstrate that DNA prepared by our modified CsCl method was also better for the transfection of Sf-9 cells than DNA prepared by the traditional CsCl method.</p>","PeriodicalId":80082,"journal":{"name":"Methods in cell science : an official journal of the Society for In Vitro Biology","volume":"24 4","pages":"155-63"},"PeriodicalIF":0.0,"publicationDate":"2002-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1023/a:1024413604663","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"22468977","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}

{"title":"Use of confocal microscopy for the study of spermatogenesis.","authors":"Margarita Vigodner,&nbsp;Lawrence M Lewin,&nbsp;Tova Glaser,&nbsp;Leah Shochat,&nbsp;Leonid Mittelman,&nbsp;Rachel Golan","doi":"10.1023/a:1024490707972","DOIUrl":"https://doi.org/10.1023/a:1024490707972","url":null,"abstract":"<p><p>Spermatogenesis consists of spermatogonial proliferation, meiosis and spermatid differentiation. Laser scanning confocal microscopy (LSCM) may be used as an advanced analytical tool to follow spermatogenesis inside the seminiferous tubules without performing histological sections. For this purpose, separated seminiferous tubules are fixed in 0.5% paraformaldehyde, stained for DNA with propidium iodide and analyzed by LSCM. By producing longitudinal optical sections in the layer of spermatogonia, spermatocytes and spermatids, stage-specific changes in their structure may be followed within the tubules by LSCM. Longitudinal z-sections may be obtained to produce three-dimensional images of the seminiferous tubules. In addition, different proteins may be followed during spermatogenesis in a stage specific manner within the tubule by incubation of the fixed seminiferous tubules with appropriate antibodies. As an example of the spermatogenesis studies using described LSCM techniques, detailed examination of spermatogonia, spermatocytes and spermatids during golden hamster spermatogenesis is presented. LSCM analysis of c-kit and SC3 protein expression at different stages of hamster spermatogenesis is demonstrated.</p>","PeriodicalId":80082,"journal":{"name":"Methods in cell science : an official journal of the Society for In Vitro Biology","volume":"24 4","pages":"169-80"},"PeriodicalIF":0.0,"publicationDate":"2002-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1023/a:1024490707972","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"22470019","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}

{"title":"Exogenous DNA expression in eukaryotic cells following microinjection.","authors":"Ann Boyd","doi":"10.1023/a:1024401924835","DOIUrl":"https://doi.org/10.1023/a:1024401924835","url":null,"abstract":"<p><p>Microinjection of nucleic acids, DNA, RNA, proteins, and any soluble material into living eukaryotic cells makes it possible to design experiments focused on single cells. In contrast facilitated transfer protocols requires hundreds of thousands of cells from which the expressed gene or intracellular effect must be detected within the culture. In addition to the immediate observable nature of the expressed product and intracellular reaction, microinjection bypasses the uptake toxicity associated with facilitated transfer of foreign material into cultured cells. The direct injection of material into the nucleus or cytoplasm allows the number of treated cells to be monitored and expression efficiencies to be observed directly. Microinjection of a hundred cells grown on small glass coverslips and subsequently counted for expression of the foreign material determines expression efficiency as a percentage of cells injected. The efficiency is based on detection of the foreign inserted gene product and does not control for relative promoter efficiency between constructs. The purpose is not to compare two constructs to each other but to monitor dual expression. The creation of marker fluorescent proteins, such as the green fluorescent protein (GFP) in the same expression plasmid with a test gene allows the immediate observation of the GFP injected cells and within the same cells the positive or negative expression of the test gene. Expression of a foreign gene, such as SV40 T antigen cloned into an expression vector can be detected four hours after microinjection of the DNA. Fusing GFP into the same expression region of the T coding sequence labels T-GFP as a fusion protein with characteristic T immunological staining nuclear patterns but allows the cells to be studied without fixation through sequential periods of observation. The direct nature of microinjection allows comparison of gene expression in a variety of cells and the determination of the number of cells expressing the exogenous material in relationship to the number of cells injected.</p>","PeriodicalId":80082,"journal":{"name":"Methods in cell science : an official journal of the Society for In Vitro Biology","volume":"24 4","pages":"115-22"},"PeriodicalIF":0.0,"publicationDate":"2002-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1023/a:1024401924835","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"22468984","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}

{"title":"Chromosome sorting and genomics.","authors":"L. Cram, Carolyn Bell, J. Fawcett","doi":"10.1007/978-94-017-0623-0_4","DOIUrl":"https://doi.org/10.1007/978-94-017-0623-0_4","url":null,"abstract":"","PeriodicalId":80082,"journal":{"name":"Methods in cell science : an official journal of the Society for In Vitro Biology","volume":"21 1","pages":"27-35"},"PeriodicalIF":0.0,"publicationDate":"2002-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"78386517","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}

{"title":"Cellular and serological markers of disease activity in Indian patients with HIV/AIDS.","authors":"S. Sehgal, U. Datta, S. Mujtaba, A. Sood, V. Vinayak","doi":"10.1007/978-94-017-0623-0_16","DOIUrl":"https://doi.org/10.1007/978-94-017-0623-0_16","url":null,"abstract":"","PeriodicalId":80082,"journal":{"name":"Methods in cell science : an official journal of the Society for In Vitro Biology","volume":"54 1","pages":"107-14"},"PeriodicalIF":0.0,"publicationDate":"2002-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"73677694","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}

{"title":"The human epithelial cell cytotoxicity assay for determining tissue specific toxicity: method modifications.","authors":"Eugene Elmore,&nbsp;Shazia Siddiqui,&nbsp;Neeraj Desai,&nbsp;Mary Pat Moyer,&nbsp;Vernon E Steele,&nbsp;J Leslie Redpath","doi":"10.1023/a:1024453300493","DOIUrl":"https://doi.org/10.1023/a:1024453300493","url":null,"abstract":"<p><p>The Human Epithelial Cell Cytotoxicity (HECC) Assay (Meth Cell Sci, 22: 17-24, 2000) has been modified to include three additional cell lines and to allow protocol adjustments for slow growing cell lines. This manuscript presents methods using human epithelial cells from ten different normal human tissues including: skin, mammary, prostate, renal, bronchial, lung, oral, ecto-cervix, colon, and liver. The HECC Assay can also be used to evaluate other types of drugs, personal care products, environmental chemicals, and potential toxicants. Human epithelial cells at an early passage are seeded into multi-well dishes. The cells are exposed to multiple concentrations of each test agent. A preliminary assay using an exposure of five days at 1 mM (if soluble) and four log dilutions is used to determine the highest concentration for the HECC Assay. In the HECC Assay, cultures are exposed for three to four days. Following the exposure period, endpoint measurements for inhibition of growth, mitochondrial function, and PCNA (proliferating cell nuclear antigen) expression or albumin synthesis (hepatocytes) are made. Data are analyzed to determine the concentration that inhibited an endpoint by 50 percent (TC(50)) for each agent in each target epithelial cell line or culture and the data are compared to determine the relative sensitivity of each epithelial cell line to the test agent.</p>","PeriodicalId":80082,"journal":{"name":"Methods in cell science : an official journal of the Society for In Vitro Biology","volume":"24 4","pages":"145-53"},"PeriodicalIF":0.0,"publicationDate":"2002-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1023/a:1024453300493","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"22468975","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}