Skip to content

Critical Steps in DNA Cell Cycle Analysis

Written by Tim Bushnell, PhD

DNA cell cycle analysis is a very powerful technique in flow cytometry. It is deceptively easy, but there are several critical things to remember to ensure successful analysis.

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. Thus, 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.

Remember 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. Thus it is essential to have a good cell count to ensure the correct amount of DNA dye is added to the sample.

Fixation choice is important.

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.

Don’t 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.

Watch the CV.

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.

Beware the doublets.

Doublets 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.

Screen Shot 2014-02-21 at 8.14.49 AM

Screen Shot 2014-02-21 at 8.15.04 AM

Control the cell cycle.

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.

Tim Bushnell, PhD

BOOKS

Advanced Microscopy

Learn the best practices and advanced techniques across the diverse fields of microscopy, including instrumentation, experimental setup, image analysis, figure preparation, and more.
flow cytometry tablet eBook cover

Modern Flow Cytometry

Learn the best practices of flow cytometry experimentation, data analysis, figure preparation, antibody panel design, instrumentation and more. 

Advanced 4-10 Color Compensation

Advanced 4-10 Color Compensation, Learn strategies for designing advanced antibody compensation panels and how to use your compensation matrix to analyze your experimental data.

Top 40 Networking Scripts For PhDs

If you want to get replies from top employers and recruiters, this ebook is for you. These networking scripts will show you the exact words ...

Informational Interviews For PhDs

If you want to learn how to set up and execute informational interviews with PhDs working in industry, this ebook is for you. Here, you ...

Industry Resume Guide For PhDs

If you have a PhD and want to create the perfect industry resume to attract employers, this ebook is for you. Here, you will get ...

Top 20 Industry Jobs For PhDs

If you want to learn about the top 20 industry careers for PhDs regardless of your PhD background, this ebook is for you. Here, you ...

Salary Negotiation For PhDs

If you have a PhD and want to learn advanced salary negotiation strategies, this ebook is for you. Here, you will learn how to set ...

Top 20 Transferable Skills For PhDs

If you want to learn the top 20 transferable skills the industry employers ranked as most important for PhDs to include on their resumes and ...

Related Posts You Might Like

We Tested 5 Major Flow Cytometry SPADE Programs for Speed – Here Are The Results

Written By: Tim Bushnell, PhD As a follow-up to our post on tSNE where we compared the speed of calculation in leading software packages, let’s ...
Read More

5 FlowJo Hacks To Boost The Quality Of Your Flow Cytometry Analysis

Written By: Tim Bushnell, PhD Primary data analysis, that is the analysis at the sample or tube level, is where the populations of interest are ...
Read More

Statistical Challenges Of Rare Event Measurements In Flow Cytometry

Written by Tim Bushnell, PhD To conclude our series on rare event analysis, it is time to discuss the statistics behind rare event analysis. The ...
Read More

How to Optimize Flow Cytometry Hardware For Rare Event Analysis

Written by Tim Bushnell, PhD “Not everything that can be counted counts and not everything that counts can be counted.” — William Bruce Cameron (but ...
Read More

How To Choose The Correct Antibody For Accurate Flow Cytometry Results

Written by Tim Bushnell, PhD Next to the flow cytometer itself, the most important component of a flow cytometry experiment comes down to the antibodies. ...
Read More

How To Achieve Accurate Flow Cytometry Calcium Flux Measurements

Written by Tim Bushnell, PhD Most flow cytometry experiments work with antibodies conjugated to a fluorochrome for some variation on immunophenotyping. However, any fluorochrome that ...
Read More