The detection of anti-nuclear antibodies (ANA) is one of the most common procedures carried out in the autoimmunity laboratory. Currently, there are several techniques that allow the detection of ANA in patient serum, such as ELISA, western-blot, immunodiffusion, radio immunoassay or indirect immunofluorescence. IFL is the major screening method used for systemic autoimmune disease diagnosis. The technique has evolved in recent years from using rodent tissue sections as substrate to using cultured tumour cells. Because the nucleus of these cells is bigger compared to that of rodent hepatocytes, ANA detection on HEp2 cell slides has enabled the discovery of many new fluorescent patterns that are associated with autoimmune diseases.
by Dr P. Munujos

The need for synchronised cell cultures
Identifying the different immunofluorescent staining patterns of HEp2 cells is often difficult due to the complexity of interpreting the images observed. In addition, the many different microscopes and reagents that can be used for autoantibody determination may have an influence on the results. The selection of antigenic substrate, developing procedure and conjugate, amongst others, is important for correct determination. Thus there is a need to combine high interpretation skills with suitable reagents; the use of high quality cell cultures is particularly important. These cell cultures must have several important characteristics, such as well spread cytoplasm, rounded and regular nuclei, a homogeneous distribution of the cell monolayer, good adherence of cells to the growing surface, absence of morphologically abnormal cells and, finally, the presence of cells in mitosis, if good results are to be guaranteed.

There are two main reasons why it is important that a certain percentage of the cells are undergoing mitosis. Firstly, some patterns are so similar that the only distinctive feature that allows them to be identified is the staining shown by the cells in mitosis. This is the case with the clumpy nucleolar pattern, where metaphase and telophase plates appear to have a fluorescent irregular ”fan-like” edge [Figure 1-A], which is described in the HEp2 glossary recommended by EULAR (European League Against Rheumatism). This characteristic helps differentiate the clumpy nucleolar pattern from other nucleolar patterns, which are not observed during mitosis. Mitosis also facilitates the identification of the coarse speckled nuclear pattern, in which speckles are condensed around the chromatin plate [Figure 1-B]. The autoantibodies anti-SSB and La, found in the serum of Sjögren’s syndrome patients, are also interesting. A fine speckled nuclear pattern is observed with these antibodies, which is difficult to identify correctly unless the fine speckles and condensation around the unstained chromatin plate can be observed in the cytoplasm of cells in metaphase [Figure 1-C]. One of the most useful staining patterns that requires cells in mitosis for its interpretation is the centromere pattern, in which fluorescent dots corresponding to centromeres line up during metaphase and telophase [Figure 1-D].
Staining of the centrioles at opposite poles of the spindle during mitosis also facilitates identification of the centriole spindle pattern [Figure 1-E].

The second important reason why it is necessary to have a percentage of observed cells undergoing mitosis is the existence of autoantibodies that react with proteins which are only expressed during certain phases of mitosis. This is the case with some spindle microtubule proteins that are involved in chromosome segregation during mitosis, proteins that have a half-life of only 60-90 seconds [Figure 1-F], [1]. The phosphoepitopes on mitotic chromosomal autoantigens (MCA) are also located exclusively on the arms of mitotic chromosomes [2]. The proportion of cells in mitosis has become an important criterion when deciding which HEp2 cell slides to purchase. In an asynchronised culture only 1-2% of cells will be in the mitotic (M) phase of the cell cycle, whereas around 10-20% of cells are undergoing mitosis in a culture that has been synchronised in the early S phase of the cell cycle (the phase when DNA synthesis commences).

Methods of cell-cycle synchronisation
There are several possible approaches that can be used to achieve a certain percentage of synchronised cells in an in vitro cell culture. These approaches can be divided into biological and chemical methods. Depending on the intended application, some methods are more appropriate than others.

Biological methods
One of the simplest ways to obtain  synchronised cells in the M phase of the cell cycle is the method known as mitotic shake-off. This method takes advantage of the fact that cells in the process of dividing  undergo cytoskeletal changes and become round, so that they are only weakly attached to the culture surface. With gentle shaking of the culture flask, mitotic cells detach and can be collected from the supernatant. These cells constitute a synchronised population, and after being seeded into a new culture flask, they will almost immediately enter into the G1 phase of the cell cycle (when they begin to increase in size).

Another method of synchronising mammalian cell cultures is serum deprivation, which arrests cells in the G0 phase of the cell cycle (the quiescent phase). The transition from the G1 to the G0 phase can be achieved by removing growth factors present in the serum from the culture medium. Once the cells are quiescent, they can be synchronised by replenishing the medium with serum, which induces them to re-enter the G1 phase. The continual dividing of the cells which follows results in a culture with a high density of cells and thus cell to cell contact. This method, known as the contact inhibition synchronisation procedure, arrests the cell cycle in the early G1 phase. The method can be combined with serum deprivation enabling better results for certain cell types.

Finally another important synchronisation method is centrifugal elutriation. This technique separates cells by size and density simultaneously. As the normal cell cycle progresses, cells increase in size from the small early G1 phase cells to G2 or M phase cells that are double the size of G1 cells. The method is based on a special centrifuge chamber in which the centrifugal force is countered by the medium flow force, causing the cells to distribute along the chamber according to their size, from where they can be collected and seeded [3].

Chemical methods
There are a number of different methods of cell cycle synchronisation that are based on the use of specific drugs that inhibit cell cycle progression (pharmacological arrest) at a certain phase of the cell cycle. The cell populations in phases beyond the point of arrest continue the cell cycle until they reach the phase of arrest. When the medium containing drug is removed and replaced by fresh culture medium, cells continue the normal cell cycle in a synchronised manner. There are several biochemical cell cycle inhibitors described in the literature, which arrest cell activity at different phases of the cell cycle. Lovastatin, daidzein, thymidine and aphidicolin halt the cell in the G1 phase [Figure 2]; hydroxyurea, 5-fluorodeoxyuridine and thymidine target the S phase; and nocodazole and colchicine inhibit the cell cycle in the M phase. However there is rapid decay following release from arrest whatever phase of the cell cycle the drug targets [4, 5].

Cell cycle inhibitors targeting the S phase are the most suitable for applications involving enrichment and visualisation of mitotic figures in a monolayer culture. Of these, aphidicolin gives high yields without noticeable perturbations or toxic effects. This drug prevents G1 cells from entering the S phase, and allows G2, M and G1 phase cells to continue the cell cycle until they accumulate in the G1/S border phase [6].

When monitoring the degree of synchronisation achieved, it can be observed that within the first two hours after drug removal, dividing cells are totally absent from the cell culture [Figure 3]. The reason is that those cells that were in phases between the G1/S border phase and the M phase at the start of the arrest have passed the stage of cell division after 13 hours of arrest, so there are now only G1/S border phase cells in the culture. The highest percentage of synchronised cells is achieved 12-14 hours after the drug removal. Advantage can be taken of this two hour period to fix the cells, so that those undergoing mitosis are available for the detection of ANAs that only react with proteins expressed during mitosis, or have a distinctive staining pattern that can only be identified in cells undergoing mitosis.

References
1. Gruber J et al. The mitotic-spindle-associated protein astrin is essential for proression through mitosis. Journal of Cell Science 2002; 115: 4053-4059
2. Gitlits VM et al. Novel human autantibodies to phosphoepitopes on mitotic chromosomal autoantigens (MCAs). Journal of Investigative Medicine 2000;48(3):172-182.
3. Davis PK et al. Biological Methods for Cell-Cycle Synchronization of Mammalian Cells. BioTechniques 2001; 30:1322-1331.  
4. Shima D et al. Partitioning of the Golgi Apparatus during Mitosis in living HeLa Cells. The Journal of Cell Biology 1997; 137(6):1211-1228.
5. Holt SE et al. Lack of cell cycle regulation of telomerase activity in human cells. Proc. Natl.Acad. Sci. USA 1997;94:10687-10692.
6. Pedrali-Noy G et al. Synchronization of HeLa cell cultures by inhibition of DNA polymerase a with aphidicolin. Nucleic Acids Research 1980; 8(2):377-387

The author
Dr Petraki Munujos,
BioSystems S.A.
Barcelona, Spain