The Cycletest™ Plus DNA Reagent Kit is very useful in detecting the distribution of cells within the different phases of cell cycle. It can be used for both
in vivo and
in vitro experimental models. The principle involves dissolving the cell membrane with a nonionic detergent, eliminating the cell cytoskeleton and nuclear proteins with trypsin, digesting the cellular RNA with an enzyme, and stabilizing the nuclear chromatin with spermine. Propidium iodide (PI), a fluorescent dye, is stoichiometrically bound to the clean, isolated nuclei which are then run on a flow cytometer equipped with electronic doublet-discrimination capability. Propidium iodide-stained nuclei emit fluorescent light primarily at wavelengths between 580 and 650 nm. In our lab we use the BD™ LSR II flow cytometer from BD Biosciences. This model contains 2 lasers, an argon and UV laser. It also has filters for both absorbtion and emission spectra generated by PI.
The resulting fluorescence histograms may be analyzed in order to detect the presence of an abnormal DNA stemline (DNA aneuploidy). Normal cells obtained from the same tissue or peripheral blood mononuclear cells (PBMCs) can be mixed with the sample in a second tube before staining and used as a reference to determine the degree of DNA content aberration. The DNA index (DI) is obtained by dividing the mode (or mean) of the relative DNA content of the abnormal G0/G1 population by the mode (or mean) of the normal G0/G1population. In our lab, the DI and coefficient of variation for each G0/G1 peak are reported in the analysis performed by BD Bioscience’s MODFIT software. This analysis can also be done using BD Bioscience’s CellQuest™, but in that case we have to calculate the DI manually.
The kit consists of 3 solutions: Solution A contains trypsin in a spermine tetrahydrochloride detergent buffer for the enzymatic disaggregation of the solid tissue fragments and digestion of cell membranes and cytoskeletons. Solution B contains trypsin inhibitor and ribonuclease A in citrate stabilizing buffer with spermine tetrahydrochloride. Solution C contains propidium iodide (PI) and spermine tetrahydrochloride in citrate stabilizing buffer. The PI stoichiometrically binds to the DNA at a final concentration of at least 125 ug/mL. It can be used for preparation of 40 samples. The whole procedure takes 30 minutes. 5x105 cells are required for processing. The cell sample is first suspended in the citrate buffer. The cells are then centrifuged at 400 g for 5 min at room temp. The supernatant is carefully decanted and 250 ul of Solution A is added to the pellet and kept at room temp for 10 min (for cultured cells) and 20 min for tissue samples. Then 200 ul of Solution B is added and gently mixed by tapping. After incubating at room temp for another 10 min or 20 min, for cultured cells or tissue samples, respectively, Solution C (200 ul) is added. After gentle mixing, it is kept in the dark at 4ºC for 10 min for cultured cells and 30 min for tissue samples. Then it is gently mixed and samples are analyzed on a flow cytometer.
My research aims to determine the mechanism involved in prostate carcinogenesis and its prevention by the administration of dietary agents. To study this, I use a mouse prostate model as well as human prostate cancer cell lines. By using this kit, I could correlate the distribution of cells in among the phases of the cell cycle in both normal, solid tissue and in malignant cells. I could also establish the mechanisms of dietary prevention, via both cell cycle arrest and induction of apoptosis by using this kit. Using this kit, we determined the sub-G1 population of cells which is indicative of apoptosis. We also used this kit to establish the ploidy in different stages of cervical carcinoma in our lab.
For analysis by flow cytometry, it is important to prepare single cell suspensions of the tissue. This is easier to do with in vitro studies. However, in the case of solid tissue, it is always difficult to generate single cell suspensions and if our test samples do not consist of single cells, the results obtained will be misleading. Without a single cell suspension, the data are generated from more that one cell thereby showing multiploidy results even if there is none. I used to face such problems; even if I was using normal tissue samples, I was getting a DNA index indicative of abnormal stemlines. Since using the Cycletest™ Plus DNA Reagent Kit from BD Biosciences, I have obtained clear-cut discrimination between normal and malignant cell populations in terms of both their ploidy status and cell cycle distribution.
I would, therefore, recommend Cycletest™ Plus DNA Reagent Kit for quick detection and reliable results.