The quantitative measurement of the DNA content of cells was one of the earliest applications in flow cytometry. It has become possible by specific fluorescent dyes which bound stoichiometrically to the double helix of nuclear DNA.

Staining Solutions for DNA Analysis of Mammalian Cells

 
The fluorescence inside the cell nucleus can easily be detected using flow cytometry. Thus, the amount of DNA for cell populations can be studied regarding: a) the cell cycle and b) the ploidy level. In growing tissue the DNA amount per cell is correlated to certain cell cycle phases of replication of chromosomes. Three different phases can be distinguished: G1, S, G2M (G1: normal DNA content = 2c, S: DNA synthesis, G2M: double DNA content = 4c and mitosis). Mammalian somatic cells are usually diploid with a specific genome size related to the number and individual size of chromosomes. This is in contrast to neoplastic cells which tend to have abnormal and aneuploid DNA contents. Differences in the genome size occur usually as chromosome abnor-malities (different karyotypes).Ploidy levels or cell cycle stages can be detected with Partec flow cytometers by high resolution DNA measurements within differences of 1% compared to the normal cellular DNA content. With the Partec FCM instruments in combination with Partec DNA reagents very low CV values (lower than 1,5 %) can be obtained for various cell samples.
 
Sample Preparation: Any kind of cultured cells, blood cells, bone marrow cells, body fluids biopsies, and needle aspirates, as well as solid tumor material may be used for flow analysis independently if the material is fresh, frozen (-20 or -80), fixed (form-aldehyde or ethanol) or even paraffin embedded. Enzymatic or mechanical disaggregation is usually required to isolate cells. The Partec reagent CyStain® starts with a nuclei extraction before staining with the fluorescent dye in order to achieve high resolution analysis.
 
Staining: DAPI (4’ 6-diamidino-2-phenylindole) is a UV dye. It emits a very bright blue fluorescent after excitation with the 366 nm line of mercury arc lamp in Partec flow cytometers. In contrast to other DNA stains like propidium iodide, DAPI bounds to A-T specific regions in minor grooves of the DNA helix. As a significant advantage no pretreatment of cells with RNase is needed with Partec DAPI reagents. Hence, stained samples are immediately ready for flow analysis.
 

Staining Solutions for Nuclear Plant DNA and Ploidy

 
In parallel to the development of dedicated instrumentation Partec pushed forward the development of own ready-to-use reagent kits and protocols for routine and high resolution analysis applicable to a wide range of different plant species. Analog to the staining of mammalian cells, plant nuclear DNA is stoichiometrically stained by DNA fluorochromes like DAPI, Hoechst dyes, propidium iodide and many others. As the DAPI fluorochrome (4’, 6-diamidino-2-phenylindole) turned out to be superior for the staining of nuclear DNA regarding the major part of applications most Partec reagent kits are based on this dye. DAPI combines fast and easy sample staining with high resolution results at low costs. In addition, due to the comparable low health risk, the handling of this fluorochrome is easier and more convenient as the handling of other DNA dyes. As long as the DNA content of plants within the same taxonomic family is concerned, the A-T specificity of DAPI does not cause ploidy errors. As result of Partec's long experience in the analysis of nuclear DNA in plants, we offer different staining kits for routine ploidy analysis (one step procedure) and high precision DNA analysis (two step procedure, different organisms), and for the absolute genome size determination.
 
Preparation procedure (fresh or frozen material): Any nuclei containing material of plants like seedlings, roots, flowers or seeds can be used. The most frequently analyzed material is leaf tissue. However, for specific applications particular tissues have to be used for the analysis i.e. microspores or pollen, embryos and endosperm or protoplasts. With all kinds of material the preparation procedure is based on the extraction of the intact nuclei and their subsequent fluorescent staining. Tissue material i.e. is prepared as follows:
 
_ take appr. 0.5 cm2 leaf or other tissue material
_ chop with a sharp razor blade in extraction buffer (or in combined extraction/staining buffer
   of CyStain® UV Ploidy)
_ Filter the nuclei suspension through a CellTrics® disposable filter directly into the sample
   tube
_ incubate 0.5 to 3 minutes (depending on the sample)
_ add staining buffer
_ analyze with UV excitation in your Partec flow cytometer or with any other flow cytometer
   with UV excitation
 
CyStain® PI absolute P follows the same procedure but requires longer incubation and treatment with RNase.