【Flow cytometry】A Review of Apoptosis Data Analysis Techniques

The concept of apoptosis and its characteristics

Apoptosis: also known as programmed cell death (PCD), is the autonomous and orderly death of cells controlled by genes in order to maintain the stability of the internal environment, which plays a necessary role in the removal of unwanted or abnormal cells by multicellular organisms, and belongs to the "suicide" behavior of cells.

Typical features of apoptosis

1. loss of mitochondrial membrane potential;
2. eversion of cell membrane PS (phosphatidylserine);
3. Nuclei condense and rupture.

Apoptosis

Programmed death is an active death process taken by the body to better adapt to the living environment.

Heat analysis of apoptosis research

Apoptosis and disease

Apoptosis hot issues

• Cells are stimulated by extracellular signals to initiate apoptosis
• Activation of intracellular apoptosis-specific proteases such as caspase
• Mitochondrial regulation of apoptosis
• Regulation of apoptosis by the Bcl-2 protein family

Stage of apoptosis

Early Events: Phosphatidylserine (PS) Valgus ØMitochondrial Damage ØCytochrome C Release	Metaphase Events: Ø Activation of Cholesterolase 3/7/8/9	Late Events: O DNA fragmentation

Apoptosis assay – Annexin V

9 key technologies

1. Seeding logarithmic growth
   cells usually does not exceed 1×106/ml to avoid causing apoptosis
2. Harvesting
   The supernatant must be collected first, centrifuged to collect the pellet, and then digest and collect the adherent cells, and combine the supernatant and digested cells.
3. Digestion Accutase
   avoids false positives caused by over-digestion of PS eversion.
   Replacing trypsin with eBioscience's Accutase enzyme has been validated in a variety of cell lines with no effect on gentle, long-term digestion of cells. (Highly recommended for experiments where apoptosis is not obvious) using cell digest - actual effect comparison
   

   

4. Positive quality control
   There is no obvious apoptosis in Multisciences, how to adjust the compensation and set the gate?
   The Synctech Apoptosis Kit is equipped with a positive quality control solution to make compensation adjustment more convenient.

   

5. Protocol: 4 tubes for single and double mixed
   staining
   • Blank
   • Annexin V
   • PI
   • Annexin V+PI

   Get on board

   • Blank
   • 50% Blank + 50% Annexin V
   • 50% Blank + 50% PI
   • 50% Blank + 50% Annexin V+PI
   • 50% Blank + 25% Annexin V+PI + 25% Annexin V
6. The voltage is large enough that it almost goes beyond the boundary.

   

7. The number of cells is large enough that all the cells are collected.
8. Compensation Horizontal and vertical: 3, 4 quadrants horizontally flat; 2. 4 quadrants are vertical
9. Beautiful picture of the golden section (rule of thirds).