Journal
JOURNAL OF CLINICAL BIOCHEMISTRY AND NUTRITION
Volume 53, Issue 1, Pages 27-35Publisher
JOURNAL CLINICAL BIOCHEMISTRY & NUTRITION
DOI: 10.3164/jcbn.12-104
Keywords
flow cytometry; cell-block; crocidolite; endocytosis; quantitative assessment
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Funding
- Princess Takamatsu Cancer Research Fund [10-24213]
- Ministry of Health, Labour and Welfare of Japan
- Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan
- MEXT Special Coordination Funds for Promoting Science and Technology Grant
- Japan Society for the Promotion of Science Fellows
- Grants-in-Aid for Scientific Research [24108008] Funding Source: KAKEN
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Exposure to asbestos fibers increases the risk of mesothelioma in humans. One hypothetical carcinogenic mechanism is that asbestos fibers may directly induce mutations in mesothelial cells. Although the uptake of asbestos fibers by mesothelial cells is recognized, methods for the quantification of the uptake have not been well established. In the present study, we evaluated two distinct methods, using crocidolite fibers and MeT5A mesothelial cells. One method is histological evaluation using the cell-block technique, which allows for the direct cross-sectional observation of cells and fibers. We found the bright field observation with x1000 magnification (oil-immersion) of the sample with Kernechtrot staining was most suitable for this purpose. The other method is flow cytometric analysis, which permits the evaluation of a much larger number of cells. We observed that the side scatter (SSC) increased with the intracellular fibers, and that the mean SSC ratio (treated/control) was useful for quantification. We could collect the cells with abundant internalized crocidolite fibers by sorting. Results of the two methodologies were correlated well in the experiments. The quantities of internalized fibers increased with incubation time and loaded dosage, but they were inversely associated with cellular density in culture.
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