Apoptosis is a form of cell death distinguished from necrosis, and cells in this state have unique properties in morphology, biochemistry and molecular biology. Apoptosis can be detected on the basis of altered cell membrane permeability, such as measuring the release of labeled proteins, labeled nucleic acids and labeled enzymes, and the entry of dyes into the cell. Currently, there are 1 main method to detect apoptosis based on altered cell membrane permeability: flow cytometry of phosphatidylserine externalization to detect apoptosis.
Principle
The basic principle of flow cytometry detection of apoptosis by phosphatidylserine externalization is that phosphatidylserine (SP) molecules are usually present only on the inner side of the cell membrane, and in the early stage of apoptosis, the PS molecules in the cell membrane are flipped from the inner layer of the lipid bilayer to the outer layer, resulting in the formation of PS externalization.
Annexin V is a Ca2+-dependent phospholipid-binding protein with a molecular mass of 35-36 kDa. It has a high affinity for PS and binds to the surface of apoptotic cells via externalized PS. Conjugated to the fluorescent dye fluorescein isothiocyanate (FITC), annexin V (annexin V FITC) also retains a high affinity for PS and can be used as a probe for flow cytometry analysis of apoptotic cells.
Operation method
Flow cytometry detection of apoptosis by phosphatidylserine externalization
Principle
The basic principle of flow cytometry detection of apoptosis by phosphatidylserine externalization is that phosphatidylserine (SP) molecules are normally found only on the inner side of the cell membrane, and in the early stage of apoptosis, PS molecules in the cell membrane flip from the inner layer of the lipid bilayer to the outer layer, forming PS externalization. Annexin V, a Ca2+-dependent phospholipid-binding protein with a molecular mass of 35-36 kDa, has a high affinity for PS and binds to the surface of apoptotic cells via externalized PS. Conjugated to the fluorescent dye fluorescein isothiocyanate (FITC), annexin V (annexin V FITC) also retains a high affinity for PS and can be used as a probe for flow cytometry analysis of apoptotic cells.
Materials and Instruments
Equipment: Move The basic process of flow cytometry for the detection of apoptosis by phosphatidylserine externalization can be divided into the following steps: A Culturing cells, apoptosis induction process as before. B Collect cells, suspended cells are collected directly. Adherent cells are first gently blown with a dropper, and the centrifuged tube of cells that have been dislodged is collected. Add appropriate amount of 0.02% EDTA (3~4 mL) to digest non-dembedded cells and collect them into the above centrifuge tubes. centrifuge at 1000 r/min for 5 min and discard the supernatant. C Wash the cells twice with cold PBS. D Filter through a 400-mesh sieve once. E Suspend the cells in 1x binding buffer and adjust the cell count to 1 x 106 cells/mL. F Take 100 μL of cell suspension into a 5 mL glass tube. G Add 5 μL of V-FITC and 10 μL of PI [Control 1: omit step (4); Control 2: add V-FITC only; Control 3: add PI only]. H Mix gently and incubate for 15 min at room temperature under light. I Add 400 μL of 1x binding buffer and analyze immediately by flow cytometry (also by fluorescence microscopy). Caveat 1 PI Harmful if inhaled, ingested or absorbed through the skin. Wear gloves and a white coat in a chemical fume hood accordingly. For more product details, please visit Aladdin Scientific website.
① Flow cytometer
② 10 mL centrifuge tube
③ Micro pipette and its tip
④ 5 mL glass tube
⑤ 400-mesh sieve
⑥ HeLa cells (or other cells).
Reagents:
① Binding buffer
② 0.02% EDTA
① Binding buffer ② 0.02% EDTA ③ PBS
① Binding buffer ② 0.02% EDTA ③ PBS
⑤ 10x binding buffer (0.1 mol/L HEPES-NaOH, pH 7.4)
(0.1 mol/L HEPES-NaOH, pH 7.4; 1.4 mmol/L NaCl; 25 mmol/L CaCl
2
(diluted to 1x before use))
⑥ VP-16 100 mmol/L (-20 °C storage)
⑦ PI 50 μg/mL (pH7.4 PBS preparation), stored at 4 ℃.