The cell cycle analysis is a robust technique in flow Cytometry. It is not daunting to perform but needs some care. Let’s highlight the mistakes that usually done by the users:

Don’t assemble sufficient events

The cell cycle analysis comprises the fitting of the data with the help of several mathematical models that depicts the behavior of the data. To have sufficient data, one should have assembled 100 events for every channel between the commencing of the G1 peak and the end of the G2/M peak. Hence, if the G1 pear begins at 50000 and the G2/M phase ends at 120000, then the dataset should include (120000-40000)*100= 500000 events.

Not selected a fixative carefully

The Cross linking agents like aldehyde can decline the dye binding because they get introduced chromatin crosslinking. The Dehydrating fixatives like methanol and ethanol are good, however, at high concentration can result in cell clumping. Moreover, dehydrating dyes can negatively affect fluorescent dyes as well if the DNA is being stained in the combination of surface maker stains. Hence, never forget to add some amount of little detergent that will help to hike up the access of the DNA dye.

Get confused about doublets for G2/M phase cells

The doublets which are passing through the cytometer in a side-by-side fashion can act as cells in the G2/M phase. It is significant to already arrange good single cell prep for DNA cell cycle analysis. Moreover, be sure to collect the pulse geometry measurements to provide that doublet discrimination gating can be executed on the sample.

Forget about the CV’s

The fact is that the CV’s of the G0/G1 peak is used to measure the quality of the DNA histogram. It can be changed with the help of flow rate and laser alignment. Moreover, the CV is better if it has evaluated at a lower rate. Therefore, it is suggested to execute DNA samples at low flow rates on a well-aligned instrument.

Missed the RNA

Some dyes (PI, for example) will bind to both DNA and RNA. If using PI, it is critical to add an RNAse to the staining buffer. Failure to do so will result in messy DNA histograms.

No control to the cell cycle itself

It will be well-appreciated to combine a DNA cell cycle control into all experiments. To indulge in it will allow for better characterization of changes in DNA cell cycle within some period of time, endows for analyzation between samples/machines/days, hence enhancing reproducibility and confidence.

In simple words, the cell cycle analysis is a robust tool in the flow Cytometry toolbox, but necessary steps should be followed to get the accuracy. Never consider adding some PI to a sample will result in good DNA histogram.

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