Cell Cycle Tutorial Contents

Experimental Requirements1. Cells in suspension2. 70% ice-cold ethanol3. PBS4. RNase5. 37 o C incubator6. DNA dye of choice e.g. PI, DAPI, 7-AAD, Hoechst 333427. Flow Cytometer, BD LSRII or BD FACSCanto IIDNA DyesThere are numerous fluorescent DNA dyes used for multiple purposes not all aresuitable for flow cytometric cell cycle analysis. Only fluorescent DNA dyes that bind toDNA in a linear manner can be used for flow cytometric analysis of the cell cycle. Thusas DNA content of a cell increases the fluorescent signal from the DNA dye increasesproportionally in a linear manner.Commonly used DNA dyes used for flow cytometric cell cycle analysis includepropidium iodide (PI), 4’,6-Diamidino-2-phenylindole dihydrocloride (DAPI), 7-aminoactinomycinD (7-AAD), ToPro-3 and Hoechst 33342. There are numerous other dyessuch as Sytox16 a green fluorescent DNA dye as well as other live cell cycle dyes, suchas DRAQ5, and the new range of dyes from Invitrogen.The common dyes, PI ex 350 & 488nm em 619nm, 7-AAD ex 488nm em 650nm,ToPro-3 ex 633nm em 660nm and DAPI ex 350nm em 461nm are for use with ethanolfixed cells only. Hoechst 33342 ex 350 nm em 461nm is the commonly used DNA dyefor live cell cycle analysis.PI is normally used at 50 µg/ml, 7-AAD at 25 µg/ml, ToPro-3 10nM, DAPI at 1µg/ml.Hoechst 33342 titrated for each cell type and has to be incubated with live cells at 37 o Cfor varying times for each cell type. A reasonable starting point for most cells is 10 µg/mlfor 45 minutes at 37 o C.