細胞週期染色檢測 | Cell Cycle Staining Kit貨號 KTA2020

 

細胞週期染色 (Cell Cycle) 研究的意義

細胞DNA含量的測量，是確定細胞週期階段(cell cycle)（G1，S和/或G2 / M）的最常見方法。是基於隨著細胞在細胞週期中，G1期的細胞具有一組成對的染色體，並且在DNA含量方面是一致的。 另一方面，DNA的數量在S期開始增加一倍，因此DNA的數量是G1數量的一到兩倍。 與G1中的細胞和兩組成對的染色體相比，G2 / M期的細胞具有的DNA量是其兩倍。Abbkine細胞週期染色試劑盒(Cell Cycle Staining Kit, KTA2020) 利用一種核染料(nuclear dye)，該核染料與細胞中的核酸結合會產生螢光信號(fluorescence signal)，該信號與細胞DNA含量(DNA content) 成正比，可方便，準確地確定細胞各相中細胞週期(cell cycle)的百分比。

 

Figure 5. Flow cytometry analyzed MCF–7 cell cycle proportions. Representative cell cycle distribution histograms showing apoptosis in MCF–7 cells treated for 72 h with lead compounds such as A (5b) and B (3a) at concentrations of 5 and 10 mM, and analysis of the number of cells at each cell cycle stage with all phases.

Product name	Cell Cycle Staining Kit
Applications notes	Abbkine Cell Cycle Staining Kit utilizes a nuclear dye, the binding of which to nucleic acids in the cell results in fluorescence signal, which is proportional to cellular DNA content, providing a convenient and accurate determination of the percentage of cells in each phase of the cell cycle.
Kit components	• Nuclear Dye (50×) • Assay Buffer (10×) • RNase A (100×)
Features & Benefits	• Flow cytometric analysis of cell cycle progression. • Ready-to-use reagents for staining cells. • The appropriate observe channel is Ex/Em=535/615 nm.
Usage notes	The optimal concentration of the Nuclear Dye and incubation time varies depending on the specific application. The staining conditions may need modified according to the particular cell type.
Storage instructions	Stable for at least 12 months at recommended temperature from date of shipment. Gel pack with blue ice.
Shipping	Gel pack with blue ice.
Background	The most common approach to determining the cell cycle stage (G1, S, and/or G2/M) is based on measurement of cellular DNA content. As cells progress through the cell cycle, cells in the G1 phase have one set of paired chromosomes and are uniform with respect to DNA content. On the other hand, the amount of DNA begins to double during S phase, so that the amount of DNA is between one and two times the amount in G1. Cells in G2/M phase have double the amount of DNA compared to cells in G1 and two sets of paired chromosomes.

(標準流程見原廠網站)

1.處理細胞依操作用手冊建議方法。
2.對於非貼壁細胞，通過離心（4oC，600g，5min）收集1-2×10⁶個細胞。對於貼壁細胞，首先用胰蛋白酶(Trypsin)消化細胞，然後離心。
3.用冰冷的PBS洗滌細胞沉澱兩次。
4.將細胞600g離心5分鐘，然後將試管轉移至冰中。
5.用冰冷的70％乙醇(70% ethanol)在蒸餾水中緩慢重懸細胞。將細胞放在-20°C持續2小時或更長時間（建議隔夜固定12-24小時）。在染色和分析之前，細胞可以在-20°C下保存數週。

注意：將細胞固定在單個細胞懸液中非常重要。細胞固定時容易結塊。非常緩慢地逐滴添加初始體積的70％乙醇同時輕輕渦旋有助於防止細胞結塊。

6.在4°C下以1000g離心5分鐘。
7.除去乙醇，並將細胞重懸於1 mL冰冷的PBS中。
8.在4°C下以500g將細胞離心10分鐘，然後除去PBS。重複步驟7和8兩次徹底除去乙醇。
9.將細胞重懸於0.5 mL染色溶液中，並於37°C孵育30分鐘 (在黑暗)。
10.用PBS洗滌細胞兩次，並用冰冷的PBS重懸。分析其中的細胞（Ex / Em = 535/615 nm）通過流式細胞儀檢測 (24小時內)。