FLOW CYTOMETRIC ANALYSIS OF DNA-REPLICATION DURING THE DIFFERENTIATION OF 3T3-L1 PREADIPOCYTES
FLOW CYTOMETRIC ANALYSIS OF DNA-REPLICATION DURING THE DIFFERENTIATION OF 3T3-L1 PREADIPOCYTES
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DOI：
10.1002/cyto.990060610
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发表时间：
1985-01-01
期刊：
影响因子：
--
通讯作者：
AHMAD, F
中科院分区：
文献类型：
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作者：
GRATZNER, HG;AHMAD, PM;AHMAD, F
为了研究前脂肪细胞系3T3-L1诱导脂肪细胞分化过程中DNA复制的刺激作用，我们采用了流式细胞术监测DNA合成。当用甲基异丁基黄嘌呤、地塞米松和胰岛素（MDI）的混合物处理该细胞系的融合单层时，它们经历至少一轮细胞分裂，随后是许多参与脂质形成的酶的重新合成。如果mdi处理的细胞在含碘脱氧尿嘧啶（IdUrd）或溴脱氧尿嘧啶（BrdUrd）的培养基中孵育，随后用这些碱基类似物的特异性抗体和碘化丙啶（PI）染色，可以监测诱导复制的动力学。在指数生长的3T3-L1细胞和用MDI刺激复制DNA的细胞之间，IdUrd结合模式和PI染色没有差异。此外，我们开发了一种流式细胞术（FCM）方法来染色脂肪酸合成酶和定位抗原在G1期。因此，我们已经证明了这种方法的可行性，通过流式细胞仪将脂肪酸合成酶等酶的产生与IdUrd和BrUrd掺入相关联。该技术应允许研究抑制脂肪细胞分化的卤代嘧啶。
We have employed a flow cytometric method of monitoring DNA synthesis in order to study the stimulation of DNA replication during the induction of adipocyte differentiation in the preadipocyte cell line 3T3-L1. When confluent monolayers of this cell line are treated with a mixture of methyl isobutyl xanthine, dexamethasone, and insulin (MDI), they undergo at least one round of cell division, which is followed by the de novo synthesis of many enzymes that are involved in the formation of lipid. If the MDI-treated cells are incubated in iododeoxyuridine (IdUrd)- or bromodeoxyuridine (BrdUrd)-containing medium and subsequently stained with antibodies specific for these base analogues and with propidium iodide (PI), the kinetics of the induction of replication can be monitored. No differences in the patterns of IdUrd incorporation versus PI staining were observed between exponentially growing 3T3-L1 cells and those that had been stimulated to replicate DNA with MDI. In addition, we developed a flow cytometric (FCM) method for staining fatty acid synthetase and localizing the antigen in the G1 phase. We have thus demonstrated the feasibility of this methodology for correlating by FCM the production of enzymes such as fatty acid synthetase with IdUrd and BrUrd incorporation. The technique should permit studies of the inhibition of differentiation of adipocytes by halogenated pyrimidines.