Document Detail

Establishment of a high content assay for the identification and characterisation of bioactivities in crude bacterial extracts that interfere with the eukaryotic cell cycle.
MedLine Citation:
PMID:  19111838     Owner:  NLM     Status:  MEDLINE    
High content microscopy as a screening tool to identify bioactive agents has provided researchers with the ability to characterise biological activities at the level of single cells. Here, we describe the development and the application of a high content screening assay for the identification and characterisation of cytostatic bioactivities from Myxobacteria extracts. In an automated microscopy assay Sf9 insect cells were visualised utilising the stains bisbenzimide Hoechst 33342, calcein AM, and propidium iodide. Imaging data were processed by the ScanR Analysis-software to determine the ploidy and vitality of each cell and to quantify cell populations. More than 98% of the Sf9 cells were viable and the culture consisted of diploid ( approximately 30%), tetraploid ( approximately 60%), polyploidic (<10%) and apoptotic (<5%) cells. Treatment with the reference substances blasticidin, colchicine, paclitaxel, and cytochalasin D induced changes in ploidy and vitality, which were characteristic for the respective bioactive substance. Furthermore, crude extracts from the chivosazole producing Myxobacterium Sorangium cellulosum So ce56 induced an increase of polyploid cells and a decrease in total cell count, while a mutant producing nearly no chivosazole triggered none of these effects. Purified chivosazole induced the same effects as the wild type extract. Similar effects have been observed for the reference compound cytochalasin D. On the basis of this assay, crude extracts of ten different Myxobacteria cultures were screened. Three extracts exhibited strong cytotoxic activities, further five extracts induced weak changes in the ploidy distribution, and two extracts showed no detectable effect within the assay. Therefore, this robust assay provides the ability to discover and characterise cytotoxic and cytostatic bioactivities in crude bacterial extracts.
Nickels A Jensen; Klaus Gerth; Tim Grotjohann; Dieter Kapp; Matthias Keck; Karsten Niehaus
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2008-12-09
Journal Detail:
Title:  Journal of biotechnology     Volume:  140     ISSN:  1873-4863     ISO Abbreviation:  J. Biotechnol.     Publication Date:  2009 Mar 
Date Detail:
Created Date:  2009-03-16     Completed Date:  2009-08-24     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  8411927     Medline TA:  J Biotechnol     Country:  Netherlands    
Other Details:
Languages:  eng     Pagination:  124-34     Citation Subset:  IM    
Department of Proteome and Metabolome Research, Faculty of Biology, Bielefeld University, Bielefeld, Germany.
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MeSH Terms
Cell Cycle / drug effects
Cell Line
Cell Proliferation / drug effects
Colchicine / pharmacology
Coloring Agents / metabolism
Complex Mixtures / pharmacology*
Cytochalasin D / pharmacology
Cytostatic Agents / pharmacology*
Drug Discovery
Fluorescent Dyes / metabolism
Image Processing, Computer-Assisted / methods*
Macrolides / pharmacology
Microscopy / methods*
Myxococcales / metabolism*
Paclitaxel / pharmacology
Pyrrolidinones / pharmacology
Reg. No./Substance:
0/Coloring Agents; 0/Complex Mixtures; 0/Cytostatic Agents; 0/Fluorescent Dyes; 0/Macrolides; 0/Pyrrolidinones; 0/blasticidin A; 0/chivosazol A; 22144-77-0/Cytochalasin D; 33069-62-4/Paclitaxel; 64-86-8/Colchicine

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