If cells are treated with something, the easiest thing to observe is whether it kills the cells or not, however, lots of treatments -especially the most interesting ones- likely have sub-lethal effects on cells that aren’t as simple to quantify.
Observing cell cycling in a cell line is an excellent- and relatively easy- way to check whether a condition impacts mitosis. Established cell lines growing well will have constant proportions of cells in the G0/G1-, S-, and G2-phases. This occurs naturally because all of the cells are genetically identical and live in the same conditions.
These sub-lethal changes in these conditions could lead to a number of concomitant adjustments as the cells react to the stimulus. Cells might turn genes on, turn genes off, secrete substances into the environment, or adjust surface receptor proteins. Or, they might just adjust their rate of mitosis, resulting in more or fewer cells in each stage.
Observing these changes can be easily done with a number of DNA dyes such as propidium iodide (PI). This chemical binds to DNA and fluoresces a red color at 636 nm, when stimulated by light at 493 nm (such as a blue laser in a flow cytometer). Because cell-cycle is defined by DNA content, the various phases can be distinguished by the amount of dye bound to each cell.
Imagine one cell taking up PI and then being analyzed by a flow cytometer. As the cell passes through the flow cell, it is struck by the laser and any PI within it fluoresces. Imagine the cell is in G1-phase (just after mitosis) and has chromosomes each including only one copy of the DNA ( i.e., one chromatid, see below on left). It fluoresces with an intensity consistent with the amount of PI staining that DNA. Now a second cell in G2-phase passes through the flow cell. It has twice the amount of DNA because the DNA in each chromosome has been copied and now contains two chromatids (see below on the right).
Here, it is important to note that the actual amount it fluoresces is arbitrary, i.e., it is dependent on the size of that organism’s genome. Humans have 23 pairs of chromosomes of various lengths, and Mendel’s pea plants have 7 pairs of chromosomes of various lengths. Therefore, the amount of DNA in a pea plant is therefore only about 1/3 of that in a human cell. However, we aren’t comparing cells from a pea plant to cells from a human, we’re only comparing cells from a human in a given cell cycle stage to other cells from a human in a given cell cycle stage. In cells going through cycling, each cell has twice as much DNA after passing through the ‘S’ Synthesis phase.
If these two cells are placed, according to their DNA content on an X axis, we see that cell 2 has twice the content.
When we do these analyses, we typically look at hundreds of thousands of cells at a time, so rather than seeing individual cells along this axis, we see many cells, where we can imagine them piling up atop one another at each stage.
Sometimes we might also catch cells in the midst of the S-phase, where they can have variable amounts of DNA depending on when in the S-phase we caught them. Here, an S-phase cell is pictured in red.
Now, we can clearly see that the tall column of cells on the left is G0/G1-phase, the shorter column on the right is G2-phase and the cells between them are in S-phase. Furthermore, a shape is emerging.
The shape to the right is very typical of cells in a normal culture. If a stimulus is added to stop cell cycle before S-phase, cells would pile up in G0/G1 as all the previously cycling cells passed through S and G2 (below only a small number of cells remain in G2, while cells in S-phase are entirely missing).
The example below from Prabha, Nagaram & Sannasimuthu, Anbazahan & Kumaresan, Venkatesh & Elumalai, Preetham & Arockiaraj, Jesu. (2020). “Intensifying the Anticancer Potential of Cationic Peptide Derived from Serine Threonine Protein Kinase of Teleost by Tagging with Oligo Tryptophan.” International Journal of Peptide Research and Therapeutics. 26. 10.1007/s10989-019-09817-3. shows what it might look like if cells are blocked from progressing out of G2 through mitosis.
In addition to cell cycling, it is also possible to visualize cells with less than the G1 amount of DNA. These cells are likely undergoing apoptosis (controlled cell death). In the example below, taken from Biesiekierski, Arne & Li, Yuncang & Xiao, Yin & Wen, Cuie. (2018). “Assessing the biocomptibility of biomaterials; A critical review of current in vitro toxicity assays, their advantages and limitations,” a new peak appears on the left representing these apoptotic cells. Further, the Y-axis illustrates how the number of cells in the remaining phases is greatly diminished as cells die.
As with any experiment, it is important to have controls so the shapes can be compared, but otherwise, it is a simple task to perform with relatively clear results.
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