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Submitted to: Methods in Molecular Biology
Publication Type: Peer Reviewed Journal Publication Acceptance Date: 2/26/2016 Publication Date: 11/5/2016 Citation: Li, C. 2016. Flow cytometry analysis of cell cycle and specific cell synchronization with butyrate. Methods in Molecular Biology. 1524:149-159. Interpretive Summary: Cell synchronization is an important method used in cell growth regulation research. Butyrate can block cells cycle, therefore can stop cell at specific stage of cell cycle. The property of the cell cycle arrest induced by butyrate was investigated. Method using butyrate to block cell cycle and to synchronize cell was described in this paper. Butyrate affected the cell cycle at a specific point. After released from butyrate, cells can progress into synchronous cycle of growth. The procedures using flow cytometry as a powerful tool to analyze cell population are also instructed in detail. Technical Abstract: Synchronized cells have been invaluable in many kinds of cell cycle and cell proliferation studies. Butyrate induces cell cycle arrest and apoptosis in MDBK cells. The possibility of using butyrate-blocked cells to obtain synchronized cells was explored and the properties of butyrate-induced cell cycle arrest were characterized. The site of growth inhibition and cell cycle arrest was analyzed using 5-Bromo-2’-deoxyuridine (BrdU) incorporation and flow cytometry analyses. Exposure of MDBK cells to 10 mM butyrate caused growth inhibition and cell cycle arrest in a reversible manner. Butyrate affected the cell cycle at a specific point both immediately after mitosis and at a very early stage of the G1 phase. After release from butyrate arrest, MDBK cells underwent synchronous cycles of DNA synthesis and transited through the S phase. It takes at least 8 h for butyrate-induced G1-synchronized cells to begin the progression into the S phase. One cycle of cell division for MDBK cells is about 20 h. The capability of flow cytometry to analyze multiple targets concurrently on a given cell has made it a powerful tool for researchers to study complex cellular processes of cell cycle regulation within phonotypical cell populations. By combining BrdU incorporation and DNA content analysis, not only can the overlapping of different cell populations be eliminated, but also the frequency and nature of individual cells that have synthesized DNA can be determined. |
