Li Congjun
Bovine Functional Genomics Laboratory, Animal and Natural Resources Institute, ARS, USDA, 20705, Beltsville, MD, USA.
Methods Mol Biol. 2011;761:125-36. doi: 10.1007/978-1-61779-182-6_8.
Synchronized cells have been invaluable in many kinds of cell cycle and cell proliferation studies. Butyrate induces cell cycle arrest and apoptosis in Madin Darby Bovine Kidney (MDBK) cells. We explore the possibility of using butyrate-blocked cells to obtain synchronized cells and we characterize the properties of butyrate-induced cell cycle arrest. 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. By combining BrdU incorporation and DNA content analysis, not only can the overlapping of different cell populations be eliminated, but the frequency and nature of individual cells that have synthesized DNA can also be determined.
同步化细胞在多种细胞周期和细胞增殖研究中具有重要价值。丁酸盐可诱导马-达二氏牛肾（MDBK）细胞的细胞周期停滞和凋亡。我们探索了使用丁酸盐阻断细胞来获得同步化细胞的可能性，并对丁酸盐诱导的细胞周期停滞特性进行了表征。使用5-溴-2'-脱氧尿苷（BrdU）掺入和流式细胞术分析来分析生长抑制和细胞周期停滞的位点。将MDBK细胞暴露于10 mM丁酸盐会以可逆方式导致生长抑制和细胞周期停滞。丁酸盐在有丝分裂后立即以及G1期的非常早期在特定点影响细胞周期。从丁酸盐停滞中释放后，MDBK细胞经历了DNA合成的同步周期并通过S期。丁酸盐诱导的G1期同步化细胞至少需要8小时才能开始进入S期。MDBK细胞的一个细胞分裂周期约为20小时。通过结合BrdU掺入和DNA含量分析，不仅可以消除不同细胞群体的重叠，还可以确定已合成DNA的单个细胞的频率和性质。
