Bookmark with More

7 Mistakes People Doing Cell Cycle Analyses Make

Visit the website

ExCyte is a professional flow cytometry consulting company. Our mission is to provide expert instruction and to help leaders in the field connect and grow. We specialize in providing education opportunities to anyone utilizing flow cytometry or imaging in their research.

ExCyte’s courses provide a complex understanding of flow cytometry, instantly improving the attendees’ knowledge in experimental design, instrumentation, compensation, data analysis, and figure preparation. Trained experts in the field of cytometry provide unbiased lecture style training in basic and advanced topics in cytometry at a reasonable cost to the researcher.

7 Mistakes People Doing Cell Cycle Analyses Make

DNA cell cycle analysis is a very powerful technique in flow cytometry. It is deceptively easy, which can result in simple mistakes.

The good news is that these mistakes are easily fixed or avoided altogether. Here are 7 mistakes that people doing cell cycle analyses commonly make and how to correct them:
1. They don't collect enough events.  

Cell cycle analysis involves fitting of the data using one of several mathematical models that describe the behavior of the data. These models make different assumptions about the S phase as well as the G1 and G2/M phases. To have enough data, one should collect 100 events for each channel between the beginning of the G1 peak and the end of the G2/M peak. So, if the G1 peak starts at 40,000 and the G2/M phase ends at 110,000, the dataset should contain (110,000-40,000)*100 = 7,000,000 events. 
2. They disregard the stoichiometry 

The concentration of the DNA dye must be sufficient so that it binds in proportion to the amount of the DNA in the cell. It is essential to have a good cell count to ensure the correct amount of DNA dye is added to the sample.
3. They don't carefully select a fixative 

Crosslinking agents like formaldehyde will lower the dye binding because they introduce chromatin crosslinking. Dehydrating fixatives like methanol and ethanol are better, but at high concentration can cause cell clumping. Dehydrating dyes can also negatively impact fluorescent dyes if the DNA is being stained in association with surface maker stains. Don’t forget, a little detergent can help improve the access of the DNA dye.
4. They forget 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.
5. They take their eyes off of the CVs.  

The CV of the G0/G1 peak is a measure of the quality of the DNA histogram. This can be affected by flow rate and laser alignment. The lower the CV the better, so it is critical to run DNA samples at low flow rates (narrow core streams) on a well aligned instrument.
6. They confuse doublets for G2/M phase cells.  

Doublets, or cells going through the cytometer in a side-by-side fashion, can masquerade as cells in the G2/M phase. It is critical to have good single cell prep for DNA cell cycle analysis. Watch the fixation steps and remember to filter the sample before running on the cytometer. Make sure to collect the pulse geometry measurements (H, W and A) to ensure that doublet discrimination gating can be performed on the sample.

7. They don't control for the cell cycle itself 

It is a good practice to include a DNA cell cycle control into all experiments. Doing this allows for better characterization of changes in DNA cell cycle over time, allows for comparisons between samples/machines/days thus improving reproducibility and confidence. The most common are chicken RBC and trout RBC. 

In summary, cell cycle analysis is a powerful tool in the flow cytometrists toolbox, but there are many optimization steps necessary for this deceptively easy assay. Don’t assume that one can add some PI to a sample and get a good DNA histogram. Choosing the best fixative for the assay, the right dye and a well behaved instrument are all critical for successful DNA cell cycle analysis.

Want To Learn More?
Join Our 1-Hour Cell Cycle Analysis Online Course with Special Guest Derek Davies