Apoptosis Induction Methods
Licia Miller Product Manager
Apoptosis is a programmed cell death process. Unlike necrosis, apoptosis is an orderly, gene-regulated, and active cell death process. Apoptosis plays an important role in the development of organisms, the maintenance of tissue homeostasis, the regulation of the immune system, and the occurrence and development of diseases. The main characteristics of apoptosis include reduced cell size, intact cell membranes but with vesicular protrusions, chromatin condensation, and DNA fragmentation.
Common apoptotic proteins include the Caspase family, such as Caspase-3, Caspase-8, and Caspase-9, which play a key role in the execution phase of apoptosis. Bcl-2 family proteins, including anti-apoptotic Bcl-2 and Bcl-xL and pro-apoptotic Bax and Bad, control the release of cytochrome c by regulating mitochondrial outer membrane permeability, thereby activating the Caspase cascade reaction. p53 protein is activated in response to DNA damage and promotes apoptosis by transcriptionally regulating downstream genes such as Bax and PUMA.
Common apoptosis induction methods can be divided into two categories: biological induction and chemical induction:
Biological induction mainly initiates apoptosis signaling pathways through the interaction between receptors and ligands on the cell surface. For example, Fas receptor activates the extrinsic apoptosis pathway and induces cell apoptosis by binding to Fas ligand. TNF receptor can also initiate apoptosis signaling pathways by binding to TNF ligand. These receptor-mediated apoptosis pathways usually involve the activation of death domain proteins (such as FADD) and Caspase-8, which in turn triggers the Caspase cascade reaction and leads to cell apoptosis.
Chemical induction is the induction of cell apoptosis by direct action of chemical drugs on key proteins or signaling pathways in cells. Commonly used chemical inducers include: Staurosporine, MG-132, hydrogen peroxide, etc. These chemicals can cause cell apoptosis through different mechanisms, such as damaging DNA, destroying mitochondrial function, interfering with cell cycle, etc.
Biological induction
This article describes a general protocol for inducing apoptosis in Jurkat cells using an anti-Fas receptor (anti-CD95) monoclonal antibody. Our mouse monoclonal anti-Fas antibody (Ab006530) can be used for apoptosis indications.
1. Culture Jurkat cells in RPMI-1640 containing 10% fetal bovine serum (FBS) in a 37°C, 5% CO2 incubator.
2. The cells were centrifuged at 300-350 × g for 5 min and the cell pellet was resuspended in fresh medium to a final concentration of 5 × 105 cells/mL.
3. Add anti-Fas (anti-CD95) mAb to the appropriate concentration and incubate in a 37°C incubator for 2-4 hours.
The reference final concentration of anti- Fas/CD95 mAb is 2-20 mg/mL.
4. The cells were harvested by centrifugation at 300-350 × g for 5 min.
5. Remove all medium and resuspend cells in PBS.
6. Repeat steps 4 and 5 once, and resuspend the cells in PBS for the second time to a final concentration of 1.5 × 106 cells/mL.
7. Detect cell apoptosis. Collect cells and select appropriate detection methods. If you want to detect specific apoptotic proteins, you can collect appropriate amount of cells, prepare lysate, and use it for experiments such as protein blotting or immunoprecipitation, and compare the apoptotic protein level with that in the control group.
Chemical induced
Apoptosis inducers act on a variety of apoptosis-related proteins to promote cell apoptosis. Depending on the selected reagent and the concentration used, apoptotic events can be detected within 8-72 hours after treatment. It should be noted that different apoptosis inducers may be suitable for different cell lines, and the treatment time also needs to be determined according to the specific situation.
1. Seed adherent cells into 10 cm2 tissue culture dishes or suspension cells into T75 flasks at a concentration of approximately 106 cells /mL.
NOTE: Prepare as many samples as necessary to ensure that induction of apoptosis can be detected at the relevant time.
Prepare one dish/flask of cells as a negative control without induction treatment.
2. Add cell damaging agents at the concentrations recommended in the table below to induce cell apoptosis.
This table recommends final concentrations of cell damaging agents that can be used to induce apoptosis via p53-dependent G1 arrest:
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3. Add an appropriate volume of buffer or solvent to the negative control.
4. An additional set of cells can be set up with the addition of a spectrum of apoptosis inducers, Z-VAD-FMK, at a concentration of 50 µM (prepare stock solution in DMSO) as a further control.
5. Harvest cells at different times, i.e., 8, 12, 16, 24, 48, and 72 hours after adding the cell damaging agent.
6. Detect cell apoptosis. Collect cells and select appropriate detection methods. If you want to detect specific apoptotic proteins, you can collect an appropriate amount of cells, prepare lysate, and use it for experiments such as protein blotting or immunoprecipitation to compare the apoptotic protein levels with those in the control group.
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