Hello Dr. Sudheer Babu

A slight deviation from 100% (like 96% or 105%) is often acceptable, especially if the experimental conditions are well-controlled and the deviation is consistent. But I feel it will be worth investigating to ensure accuracy.

If the percentage is less than 100%, investigate whether a significant portion of cells are in G0 or have died as they will not be in G1, S, or G2 and are often excluded from cell cycle analysis. You may use markers to distinguish between cycling and non-cycling cells. Proteins such as PCNA, which is involved in DNA replication, and Ki-67, a marker of cellular proliferation, may be used to identify cycling cells. Conversely, markers like those associated with cellular senescence can help identify non-cycling cells.

Cells undergoing apoptosis or necrosis may have degraded DNA and appear with a DNA content lower than G1, thus contributing to the percentage that is missing. If there is a significant sub-G1 population, you might need to analyze and exclude it from the main cell cycle analysis.

Cellular debris and doublets can falsely inflate the DNA content measurement. If these doublets or cell aggregates are not properly excluded during analysis, it can lead to an overestimation of DNA content and misinterpretations of the cell cycle phases. Pass the cells through a filter before analysis to remove clumps and debris. Use parameters like forward scatter height (FSC-H) vs. forward scatter area (FSC-A) or side scatter height (SSC-H) vs. side scatter area (SSC-A) to gate out doublets. These parameters help distinguish single cells from cell clumps (doublets) because doublets will have a different relationship between height and area compared to single cells.

If cells are arrested at a particular phase of the cell cycle, it can lead to an apparent increase in the percentage of cells in that phase. If you suspect cell cycle arrest, consider using technique like BrdU labelling to differentiate between cells actively synthesizing DNA (S-phase) and those arrested in S-phase.

You may have to consider other factors as well such as whether your experiment (say drug treatment) may be affecting the cell cycle and causing shifts or arrests. Ensure that FlowJo’s cell cycle analysis parameters are correctly configured and optimize the scale of your DNA content histogram to better visualize the peaks and valleys of G1, S , and G2/M phases.

Best,