How to detect apoptosis by flow cytometry

Apoptosis, usually called programmed cell death, is an active cell death process controlled by genes. More and more data have found that apoptosis is closely related to many diseases, such as cancer cells have the ability to continue to proliferate and resist apoptosis. The analysis of apoptosis by flow cytometry can provide an important reference index for tumor diagnosis, curative effect evaluation and prognosis prediction. However, many experimental students often encounter too many cell deaths but fail to detect apoptosis, and the repeated results are inconsistent. This article will take a look at how to make the data of apoptosis more beautiful and accurate!

First, make clear the difference between apoptosis and necrosis.

Apoptosis is an active cell death event involving the activation, expression and regulation of a series of genes. It is the death process of cells actively striving for better adaptation to the environment. The specific manifestations are: budding to form apoptotic bodies, DNA cleavage between nucleosomes, phagocytosis and digestion of apoptotic bodies, etc. [1] (as shown in the following figure).

Fig. 1: the process of apoptosis [1]

Necrosis is a passive process, which is a death process in which cells are subjected to strong physical and chemical or biological factors, leading to disorderly changes in cells, that is, self-injury in pathological state. The specific manifestations are: cell swelling, cell membrane rupture, cell contents overflow, slow cell nucleus change and insufficient DNA degradation, which will cause serious local inflammatory reaction (as shown in the following figure).

Fig. 2: Process of cell necrosis [1]

First, the principle of flow cytometry.

Annexin V and PI double staining method is a classical method for flow detection of apoptosis. Its principle is that phosphatidylserine (PS) on the cell membrane flips from the inside of the cell membrane to the surface of the cell membrane at the early stage of apoptosis (Figure 3 below).

Fig. 3: PS turned out from the cell membrane in the early stage of apoptosis.

This method is simple, rapid and accurate to distinguish living cells, apoptotic cells and necrotic cells. The specific principle is as follows:

Therefore, when annexin V binds to PI, it can be used to identify living cells, apoptotic cells and dead cells.

Second, the experimental operation steps

Third, data analysis.

Annexin V-FITC/PI apoptosis detection kit uses FITC labeled Annexin V as a probe. The maximum excitation wavelength of FITC is 488 nm and the maximum emission wavelength is 525 nm. The green fluorescence of FITC was detected in FL 1 channel. The maximum excitation wavelength of PI-DNA complex is 535 nm, and the maximum emission wavelength is 6 15 nm. The red fluorescence of PI was detected in FL2 or FL3 channel. Through software analysis, with FITC as abscissa and pi as ordinate, a two-color scatter chart is drawn.

As shown in the figure below; Cells can be divided into four quadrants;

Figure 4: Cell distribution in four quadrants.

For example, it is usually used to study the effects of drugs or genes on cell apoptosis. By comparing the control group with the drug group, it was found that the compound could induce the apoptosis of prostate cancer cell line PC-3M. Compared with the control group (0μM), the proportion of late apoptotic cells in the treatment group (20μM) increased from 1.79% to 1 1.39%. In addition, the percentage of living cells, apoptotic cells and necrotic cells can be calculated according to the data of each quadrant.

Figure 5. Drug concentration gradient-dependent induction of PC-3M cell apoptosis [2]

IV. Frequently Asked Questions

1. How to avoid false positive results?

2. Why do you need to collect cell supernatant?

Because apoptotic cells may fall off the wall and be suspended in the culture medium, it is necessary to collect supernatant, centrifuge and collect precipitate, and combine cells digested by trypsin, otherwise the proportion of apoptosis detected may be reduced.

3. Why do you need to go to the computer for testing within 1h after dyeing?

Because apoptosis is a rapid process, it is suggested to analyze the samples within 1 hour after staining. PI is greatly influenced by time, because labeling PI will increase cytotoxicity, especially when detecting early apoptosis. If the time is prolonged, the difference of cell grouping on flow cytometry will increase obviously, so it is suggested to detect it within 1 hour.

4. Other preventive measures

References:

[1] elmore S. apoptosis: a review of programmed cell death. Toxic substance Pathol 200735:495-5 16.

Qin Min, Peng Sheng, Liu Ning, et al. LG308, a new compound with microtubule activity against prostate cancer cells, has effective anti-tumor activity [J]. Journal of Pharmacology and Experimental Therapeutics, 2015,355 (3): 473-483.