Induction of apoptosis in cells

Procedure for biological and chemical induction of apoptosis in cells.

Last edited Tues 14 Feb 2023

Apoptosis may be induced in experimental systems through a variety of methods. In general, methods to induce apoptosis can be divided into two categories: biological induction and chemical induction.

Stage 1 - Apoptosis induction

Activation of Fas or TNF receptors by their respective ligands, or by cross-linking with an agonist antibody, induces apoptosis of Fas- or TNF receptor-bearing cells. Here we describe a general protocol to induce apoptosis using an anti-Fas receptor (anti-CD95) monoclonal antibody (mAb) in Jurkat cells. Our mouse monoclonal anti-Fas antibody [UT-1] (ab11881) can be used for apoptosis indication.

Grow Jurkat cells in RPMI-1640 containing 10% fetal bovine serum (FBS) in a humidified 5% CO2 incubator at 37°C.

Harvest exponentially growing cells at a concentration of 105 cells/mL by centrifugation at 300–350 x g for 5 min.

Resuspend cells in fresh medium to a final concentration of 5 x 105 cells/mL.

Add anti-Fas (anti-CD95) mAb to the appropriate concentration. Incubate for 2–4 hr in a 37°C incubator.

For a negative control, incubate untreated cells (without anti-Fas/CD95 antibody) under the same conditions.

Apoptosis inducers act on several apoptosis-related proteins to promote apoptotic cell death. Depending on the agent selected and the concentrations used, apoptotic events can be detected between 8–72 h post-treatment. However, not all reagents will affect a particular cell line in the same way.

These are general guidelines for inducing cellular damage with chemical agents that will lead to apoptosis.

Set up your cells for treatment

Add cellular damaging agents at the recommended concentrations to induce apoptosis.

Agent
Concentration
Doxorubicin hydrochloride (ab120629)
0.2 µg/mL (25 µg/mL stock prepared in H2O)
Staurosporine (ab120056)
1 µM (1 mM stock prepared in DMSO)
Etoposide (ab120227)
1–10 µM (1 mM stock prepared in DMSO)
Camptothecin (ab120115)
2–10 µM (1 mM stock prepared in DMSO)
Paclitaxel (ab120143)
50–100 nM (stock prepared in DMSO)

Add an appropriate volume of buffer or solvent to the negative control.

As a further control, inhibitors to the different pathways can be included:

Agent
Concentration
Z-VAD-FMK (ab120382)
50 µM (stock prepared in DMSO)

Stage 2 - Harvesting cells

Harvest the cells by centrifugation at 300–350 x g for 5 min.

Remove all medium and resuspend cells in PBS.

Repeat centrifugation step and resuspend cells.

Proceed to detect apoptosis using your method of choice.

Harvest cells at different times, i.e. 8, 12, 16, 24, 48, 72 hours after the addition of the cellular damaging agent.

Proceed to detect apoptosis.

Stage 3 - Detect apoptosis

Collect cells and detect apoptosis using your method of choice.

Collect all cells.

Prepare cell lysates for either western blot detection or immunoprecipitation using your method of choice.

Always compare levels of apoptotic proteins with control-treated cells to confirm induction.