Evaluation of two distinct methods to quantify the uptake of crocidolite fibers by mesothelial cells

Abstract

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 ×1000 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.

Receptor role of the annexin A2 in the mesothelial endocytosis of crocidolite fibers

“…We previously showed that flow cytometry is useful for the quantitative assessment of crosidolite uptake by mesothelial cells based on the rationale that SSC is dependent on the presence of intracellular structures not excluding some membranous ones that change the refractive index of light. 30 The results of the current study suggest that SSC would be useful for the quantitative assessment of asbestos uptake by mesothelial cells not only for crocidolite but also for chrysotile and amosite, although we cannot compare the different types of fibers because of their differences in shape and property. In flow cytometric analysis, we have to consider minimizing the Annexin A2 in mesothelial endocytosis K Yamashita et al time lag among samples because SSC may slightly increase over time.…”

“…In addition, we observed that crocidolite fibers with a width o0.1-0.2 mm were engulfed by mesothelial cells in a morphologically similar manner to that of fiber bundles wider than 0.5 mm. 30 Therefore, the endocytosis of asbestos fibers is suggested to be within a category of phagocytosis. Distinct from professional phagocytosis by monocytic/macrophage lineages and neutrophils, phagocytosis without triggering the immune response is called non-professional phagocytosis.…”

“…30 To prepare a cell-block sample, we centrifuged the cells that had been resuspended in the medium at 800 r.p.m. (120 g) for 5 min and replaced the supernatant liquid with 10% neutral-buffered formalin (NBF).…”

“…30 Briefly, a BD FACSCalibur Flow Cytometer (BD Biosciences, San Jose, CA, USA) equipped with a 15-mW air-cooled argon ion laser excitation light source (488 nm) was used. Data acquisition and analysis were performed using Cell-Quest software (BD Biosciences).…”

Role of hemoglobin and transferrin in multi‐wall carbon nanotube‐induced mesothelial injury and carcinogenesis

“…We added MWCNT to the ferristatin II‐treated or non‐treated RPMC at a concentration of 10 μg/cm 2 . After 24 h of incubation, the amounts of MWCNT taken up by cells were calculated by flow cytometry, as described previously …”