Cell Cycle Assay Kit (Green Fluorescence)
中文名称：细胞周期检测试剂盒(绿色荧光)
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- 应用： 细胞周期
| 检测原理 | 细胞周期（cell cycle）是指连续分裂细胞从一次有丝分裂结束到下一次有丝分裂结束所经历的整个过程。在这个过程中，细胞遗传物质复制并加倍，且在分裂结束时平均分配到两个子细胞中去。细胞周期又可以分为间期（interphase）和有丝分裂期(M phase)，细胞间期又常划分为休眠期（G0），DNA合成前期（G1），DNA合成期（S），DNA合成后期（G2）。DNA周期检测可用来反应细胞周期的各个期的状况，即细胞增殖状况。利用细胞内DNA能够和荧光染料（如Cycle Green）结合的特性，细胞各个时期其DNA含量不同从而结合的荧光染料不同，流式细胞仪检测的荧光强度也不一样可检测细胞不同周期。
细胞发生凋亡时，由于胞浆和染色质浓缩、核裂解，产生凋亡小体，使细胞的光散射性质发生变化。在细胞凋亡的早期，细胞前向光散射能力显著降低，侧向光散射能力增加或没有变化。在细胞凋亡的晚期，前向和侧向散射的信号均降低。因此可通过流式细胞仪测定细胞光散射的变化观察凋亡细胞。
Cycle Green染色后，假设G0/G1期细胞的荧光强度为1，那么含有双份基因组DNA的G2/M期细胞的荧光强度的理论值为2，正在进行DNA复制的S期细胞的荧光强度为1~2之间。凋亡细胞由于细胞核发生浓缩以及DNA片段化(DNA fragmentation)导致部分基因组DNA片段在染色过程中丢失，因此凋亡细胞Cycle Green染色后呈现明显的弱染，即荧光强度小于1，在流式检测的荧光图上出现所谓的sub-G1峰，即凋亡细胞峰。 |
| 检测方法 | 荧光;流式 |
| 样本类型 | 细胞 |
| 检测时间 | 3 hours |
| 检测仪器 | 流式细胞仪 |
| 荧光 | Cycle Green |
| Ex/Em | 500/530 |
| Channel set | FITC |
| 自备试剂 | 无水乙醇，PBS(E-BC-R187) |
| 保存温度 | 该产品可在-20°C避光条件下保存12个月。 |
| 有效期 | 12个月 |
| 运输条件 | 冰袋 |
| E-CK-A211 | Annexin V-FITC/PI Apoptosis Kit | 200Assays , 100Assays , 50Assays , 20Assays |
| E-CK-A301 | Mitochondrial Membrane Potential Assay Kit (with JC-1) | 100Assays , 50Assays , 20Assays |
| E-CK-A320 | One-step TUNEL In Situ Apoptosis Kit (Green, FITC) | 100Assays , 50Assays , 20Assays |
| E-CK-A362 | Enhanced Cell Counting Kit 8 (WST-8/CCK8) | 500Tests , 100Tests , 500Tests×20 |
Q1:What is the purpose of fixation in cell cycle assay?
The purpose of fixing cells is to preserve the original structure of tissues and cells as complete as possible, avoid degradation, autolysis, corruption, and deformation of tissues and cells, deactive various enzymes in cells and tissues to prevent various molecular degeneration and dissociation, and enable cell chemicals and enzymes to be accurately located. And in the future processing and production process will not change and damage. At the same time, fixation can also make each part of the cell easy to be stained, suitable for observation, long-term preservation and analysis.
Q2:What is the concentration of absolute ethanol in the cell cycle kit? Can I also use 70%? Is it able to detect sub-G1 signal?
Absolute ethanol is anhydrous ethanol which is 100%. It is not recommended to be replaced by 70% ethanol in order to match the best condition of assay. If there is a sub-G1 spike, it can be detected. Due to nucleus concentration and DNA fragmentation, some genomic DNA fragments of apoptotic cells were lost during the staining process. Therefore, apoptotic cells showed significantlly weaker staining after PI staining than that of the normal cells, that is, the fluorescence intensity was less than 1. The so-called sub-G1 peak, or apoptotic cell peak, appeared on the flow detection result map.
Q3:Should it use serum medium or serum-free medium when opetaing cell cycle assays?
Serum-free medium is necessary for cell cycle assays. Before the start of the experiment, cells should be cultured in serum-free medium for 12h to synchronize the cycle (pay attention to cell status, do not culture cells without serum for too long, or the cell state may be too poor to proliferate, end up with shrunk G2 phase or even sub-G1 apoptotic peak). Then normal medium was used for drug treatment, intervention and other experiments, and finally samples were collected for cell cycle detection.
