Analysis of cell cycle kinetics is a critical parameter for a wide range of research areas; accuracy and consistency is of utmost importance. The Cellular DNA Flow Cytometric Analysis Kit provides a tool designed for cell-cycle analysis using flow-cytometry techniques. The kit utilizes propidium iodide (PI) to assess cellular DNA content. The PI intercalates into the major groove of double-stranded DNA excluding single-stranded nucleic acids without any preference for purine or pyrimidine base pairs. This dye binds stoichiometrically and produces a high intensity fluorescent adduct (above 600 nm) to be used as an indicator of cellular DNA content of that cell. Since PI can also bind to double-stranded RNA, it is necessary to treat the cells with RNase for optimal DNA resolution. The fluorescence distribution produced by a cell population provides a representation of the cell-cycle distribution.
The biggest advantages of the kit are its sensitivity and simplicity. The kit contains ready to use PI and RNase. The RNase supplied with the kit is guaranteed to be DNase-free so that the undesired degradation of cellular DNA is eliminated. There is no need to reconstitute any reagent like with the old fashioned method. This kit can be utilized for cell cycle analysis and to study cell populations with aberrant DNA content (aneuploidy). The kit is recommended for use with cells from solid tissues, solid tumors, and cells grown as monolayers or in suspension. Personally, I have used this kit for cell cycle analysis using various human (Mo7e, K562, HEL) and murine (Baf3, 32D) suspension cell lines as well as for mononuclear cells from patient samples. The purpose of my study was to analyze the effect of various kinase inhibitors on different stages of the cell cycle.
The protocol is easy to follow and highly sensitive. The preparation of the sample is really very easy and large number of samples can be processed simultaneously. Briefly, first make the single cell suspension (1 x 105 -1 x 106 cells) in 200 µl PBS. To quantify cellular DNA content, permeabilize the cells by fixation with 70% ethanol for 30 min at 4°C. (Samples can be stored in 70 % ethanol at -20oC for several weeks prior to PI staining and flow cytometric analysis.) If cellular DNA quantification needs to be performed on the same day, after permeabilization, wash the cells in PBS and resuspend in 500 µl of PBS. Add 2.5 ìl RNase (DNase-free) to the cell suspension and incubate at 37°C for 30 minutes. Chill the cell suspension on ice to 4°C; add 50 µl PI to the cell suspension. After PI addition, samples need to be kept in the dark at room temperature for 30 min. The samples are then ready to be analyzed by flow cytometry. I have analyzed samples on FACSCalibur™ (Becton Dickinson) using CellQuest software to determine DNA content. The relative percentages of cells in G1, S, or G2/M phase were calculated from FL-2 histograms using ModFit LT software.
The reagents are stable when stored at 2 to 8°C and shielded from light. The reagents supplied in the kit can be easily used for 200 reactions but the volumes can be easily scaled down to be used for 400 reactions. My only precaution: I recommend that before PI staining, you make sure that your flow cytometer is working properly because after PI staining, data needs to be collected on the same day. Samples cannot be stored for a long time. Sometimes reactions still work well following overnight storage at 4°C, but accuracy might be compromised.
Overall, the Cellular DNA Flow Cytometric Analysis Kit provides a highly convenient method for cell cycle analysis. The kit is economical, easy to use and comes with enough reagents to analyze a minimum of 400 samples. I would strongly recommend it as a very useful tool for cell cycle analysis.
Post-Doctoral Fellow
Oregon Health Sciences University