At cell cycle, cells have can be at different phase and it can be detected by different methods. We studied on SAOS-2 cells. SAOS-2 cells are cancerous cells in bone narrow (1). Our object was to detect number of the cells and phase at cell division by using flow cytometer with fluorescence dye in this experiment.
A eukaryotic cell has a life cycle named as cell cycle. This cell cycle has some stages in a life of a cell. These are G1 (gape 1), S (synthesis), G2 (gape 2) and M (mitotic) phase. Growth and development is in G1. Basic organelles grow and proteins production increases at G1. S phase refer synthesis phase. DNA replication and centrosome duplication occur at S. G2 is a totally preparing stage for cell. Also DNA damage control is carried out in this phase. Finally, at M phase, cell initiates to cell division. Karyokinesis and cytokinesis occur at this phase or other names respectively are nuclear and cytoplasmic division. All these phases form a cell cycle. (2)
Flow cytometer is a counting technique that cells are counted as their size, granularity, and markers on surface while flowing in a stream by a laser device. (3) This selective separation depends on where the fluorescent dye is linked. Fluoresce-activated cell sorting (FACS) is a specialized flow cytometer method (4). Characteristic fluorescence dye for a specific region of cell is used for sorting cells one by one by using laser detection device. FACS can be applied for some analysis such as cell cycle analysis, chromosome analysis or ploidy analysis, cell sorting, cell phenotypic, apoptosis, functional analysis.
Cell Cycle Analysis
In cell cycle, cells have different amount DNA content at each step. PI (propidium iodide (PI) or 4′, 6’-diamidino-2-phenylindole (DAPI) is used for staining to DNA at this analysis. These stains are detected by the laser according to the amount of DNA and then the cells are sorted accordingly. Cells are passed through this separation process depending on the amount of DNA they have in this method, and this method creates a histogram chart. It is understandable how many cells are in this phase. But this PI binds to the DNA molecule as well as to the double stranded RNA. As in this experiment, the RNA in the medium was destroyed using the RNase to prevent it. Permeabilization process is so necessary for this analysis. The stain must be inserted into the cell before analysis. Some commonly used materials such as detergents or hypotonic treatment or by solvent fixation (As in this experiment, ethanol) may be used to increase cell permeability. All cells at G1 phase have same DNA quantity and these G1 phase cells are detected by laser as they pass through the cell in a single row. One share peak on the histogram shows how many G1 phase cells are counted. The g1 peak in a normal plot is quite large and the first splash shows the cells in the g1 phase. Between G1 and G2, S peak is found and the lowest part according to G1 and G2. (5)
Material and Methods:
The experiment was initiated using previously prepared cell culture and procedure was applied in hood to make sterilized area.
Firstly, medium was removed from plate and then the cells were subjected to wash with PBS. 1 ml of Trypsin enzyme was added into cell culture and the cells were incubated for enzyme activity for 5 min. Cells were investigated whether they separate from bottom of plate or not under microscope. Cell clusters were swimming on the media. The cell clusters were taken from the plate by micropipette and placed into 1.5 ml Eppendorf. The cells in Eppendorf was centrifuged at 1200 rpm for 5 min. After centrifuging, supernatant was removed. 1 ml cold PBS was used by adding into Eppendorf by 500µl micropipette to dissolve pellet. Ethanol (-200 C) was added to pellet while vortexing gently. Cells were placed at -200 C until next week.
Cells taken from freezer were centrifuged at 1200 rpm for 10 min. Supernatant was detracted from tube and 5 ml of cold PBS was added. Centrifuging (at 1200 rpm and for 10min) and removing were repeated. Pellet was suspended by 1 ml PBS/0.1%TrintonX. For removing RNA molecules, 100µl of 200µg/ml RNase-A was added and solution was waited for 30 min at 370C for enzyme activity. As dye, 100µl of 1mg/ml PI was added. The sample was waited in dark for 10 min at RT. The sample was used at flow cytometer at 600nm-610nm filter interval.
Phases: G1 S G2
% of number of cells: 37 35 28
Table 1- The results showing % of number of cells at different phases of cell analysis by flow cytometer
We aimed to obtain number of the cells and phase at cell division by using flow cytometer with fluorescence dye. In our group, SAOS-2 cells were used for this experiment. SAOS-2 cells are a kind of cancerous cells in bone. These cells are cancerous cells and therefore have a high rate of division and are smaller than other cells.
PBS worked as a buffer solution. It formed a stable pH value by detract contents from plate. Trypsin was an essential material for this experiment. It was used for prevent the interaction of the cells with the floor of dish and between the cells by disrupting proteins on surface of the cells. Ethanol was used to introduce the dye into the cell. Ethanol can do that by forming pores on membrane. Ethanol can additionally help by fixing the cells. At the second week, The TrintonX disintegrated the cell membrane and increased the permeability of the cell pores so that the RNase could penetrate. PI, Propidium Iodide, is a fluorescence dye to stain DNA and double stranded RNA. The function of the RNase enzyme is to remove the RNA content and to allow the DNA to be stained only as the molecule we are interested in.
When the results are examined, it is seen that these are different from our expectations. We need to see a big peak in the G1 phase, almost twice as much as the last one, and a second big peak in the cells in the G2 level. Although there is more cells percent in the G1 phase, there is not a sufficient peak; on the other hand, the cell ratio is higher than expected in the s phase. Since cells are in the form of cancer cells, we do not see such a result because of the stratified stop of division. Also it is unlikely that the device is faulty or defective because the results of other experimental groups gave a closer measurement than expected. A few things may have caused this. One of them, the lack of a sufficient percentage of cells in the G2 phase may suggest that doublet populations may be causing us. Some cells can be as doublets. These doublets cannot be correctly read through the flow cytometer and are perceived as a single cell, which can cause changes in the results. The trypsin enzymes may be expected to disrupt the interactions while using trypsin, a failure in the waiting period or in practice may have formed doublets. Another reason may be that if the excess in phase s is observed, the cells have just begun the transition phase to the division phase. The cells may have been caught during the production of the molecules necessary for cleavage. And for this reason we may be able to find the cells in different phases due to the preparation of the cells or due to the delay in the timings, especially the waiting intervals in the procedure. Lastly, Contamination may also have caused a fault. External organisms such as bacteria or fungus may have settled into the petri dish, or a material that is likely to affect the cells may be misrepresenting cell phases too much or incorrectly.
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