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The cell cycle, being directly responsible for cell proliferation, is a major target for various therapies. Cell cycle analysis is therefore an important tool for studying not only how cells proliferate, but also the therapeutic mechanisms underlying its regulation. The classic cell cycle assay requires cells to be pre-fixed with ethanol and treated with RNase, before finally being stained with DAPI or propidium iodide and analyzed. Depending on the cell line being used and the needs of the experimenter these extra steps can take anywhere from several hours to an entire day. However, the Cell Cycle Assay Kit (Red) (Product No. C3851) uses a special surfactant to permeabilize the cell membrane just enough for a DNA-specific fluorescent dye (7-AAD, λex, max = 549nm, λem, max = 648nm) to enter without DNA leaving the cell. In this way, analysis of the cell cycle can be performed without pre-fixation. Just collect your cells-of-interest, incubate with the included buffer / dye for as little as 15 minutes, and measure.
Application: Cell cycle observation of HeLa cells
- Dissolve the included 7-AAD in 100 µL of DMSO (not included).
- Combine 1 µL of the 7-AAD solution from step 1 with 200 µL of the included cell staining buffer.
- Trypsinize HeLa cells and wash once with PBS.
- Collect 5.0x104 cells in a tube. After centrifuging and aspirating the supernatant, add the solution from step 2, and incubate for 15 minutes.
- Perform cell cycle analysis on a flow cytometer (†).
(† Laser wavelength = 488 nm, Filter wavelength/bandwidth = 700 nm/54 nm)
Figure. HeLa cells from the same population were stained using the indicated method, and analyzed on a flow cytometer.
(a) Cells stained with C3851. (b) Cells stained using the traditional ethanol fixation and PI/RNase method.
Both methods were able to cleanly resolve the G0/G1, S, and G2/M peaks.
Reference
- New use for an old reagent: Cell cycle analysis of DNA content using flow cytometry in formamide treated cells
