Cell cycle analysis by quantitation of DNA content was one of the earliest applications of flow cytometry. The DNA of mammalian, yeast, plant or bacterial cells can be stained by a variety of DNA binding dyes. 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.
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. The appropriate observe channel is Ex/Em=535/615 nm.
1. It is very important that the cells are fixed into a single cell suspension. Cells can tend to clump during fixation. Very slow, drop-wise addition of the initial volume of 70% ethanol while gently vortexing helps prevent cells from clumping.
2. 70% ethanol should not be made with PBS as this causes protein precipitation on fixation. Use 70 parts absolute ethanol to 30 parts distilled water.
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