A single cell tracking system in real-time.

We describe here a novel real-time cell tracking system which can measure cell migration routes under cell culture condition. This system includes a mini incubator which controls temperature and CO(2) gas flow and a PDMS (polydimethylsiloxane) chip for chemotaxis measurement. The main differences from previous ones are real-timely long-term (24h) tracking for single cell quantitatively, simple and inexpensive constitution of optical parts for illumination and imaging, and compatible to commercial well plate. The tracking principle is to trace cell images for each 0.2s by converting the live cell images to binary images of black and white. Migration results of HUVEC and NCI-H23 cells are obtained respectively using this system. The results are single cell path (x, y) during migration, cell size, migration distance, migration speed, real-time pictures and so on. This system is applicable to all kinds of researches related to cell migration such as cell angiogenesis, chemotaxis, and moreover cancer metastasis.

Green fluorescent protein (GFP) as a direct biosensor for mutation detection: elimination of false-negative errors in target gene expression.

A new method was developed to determine the mutagenic efficacy of a suspected mutagen by employing green fluorescent protein (GFP) as a direct biosensor for mutation detection. Alterations in target gene (AcGFP1) expression after mutagen [(+/-)-7p,8a-dihydroxy-9a,10a-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE)] treatment were measured to detect the mutagenic efficacy of the carcinogen. In contrast to mutagen treatment of the entire plasmid or cell culture, the target AcGFP1gene devoid of the plasmid backbone was exposed to BPDE (10-500 microM) to eliminate the need for an additional fusion gene. Shuttle vectors (pAcGFP-N1) were religated to the AcGFP1 gene with BPDE adducts (0-8.59 microM) and replicated in the eukaryotic host. This approach eliminated false-negative errors in target gene expression that arose from BPDE adduct formation in the residual plasmid backbone rather than in the AcGFP1 gene. Determination of the BPDE-AcGFP1 adducts allowed the quantitative mutagenic effect of the BPDE adducts on AcGFP1 gene expression to be monitored. The results obtained with flow cytometry and confocal microscopy validate our method and demonstrate efficient and direct use of GFP as a biosensor for mutation detection.