Staurosporine is a compound derived from the bacterium Streptomyces staurosporeus. In cell biology, it is used to trigger apoptosis, or programmed cell death. Apoptosis is a natural process that cells use to eliminate themselves in a controlled manner, which is important for tissue development and removing damaged cells. This article provides a standard protocol for using staurosporine to induce apoptosis. Researchers use this method as a positive control in experiments studying cell death to ensure their detection methods are working correctly.
Staurosporine’s Mechanism for Inducing Apoptosis
Staurosporine functions as a potent, but non-specific, inhibitor of protein kinases. Kinases are enzymes that add phosphate groups to other proteins, a process called phosphorylation, which acts as a switch for cellular activities. By broadly blocking these enzymes, staurosporine disrupts numerous signaling pathways required for cell survival.
This widespread disruption of cellular signaling activates the intrinsic pathway of apoptosis, which is centered around the mitochondria. The inhibition of survival signals leads to a shift in the balance of the Bcl-2 family of proteins. Pro-apoptotic members of this family, such as Bax and Bak, become dominant and cause the mitochondrial outer membrane to become permeable.
Once the mitochondrial membrane is compromised, it releases factors like cytochrome c into the cell’s cytoplasm. The release of cytochrome c initiates the formation of the apoptosome, a protein complex. This structure then activates a cascade of enzymes known as caspases, starting with caspase-9, which in turn activates executioner caspases like caspase-3. These executioner caspases are responsible for systematically dismantling the cell.
Experimental Preparation and Reagents
Staurosporine is supplied as a powder and must be dissolved to create a concentrated stock solution. The most common solvent for this is dimethyl sulfoxide (DMSO), as staurosporine is not readily soluble in water. A common stock concentration is 1 millimolar (mM). To prevent degradation from repeated freeze-thaw cycles, this stock solution should be divided into single-use aliquots and stored at -20°C in the dark.
Cells should be seeded into appropriate culture vessels, such as 6-well or 96-well plates, depending on the planned analysis method. The seeding density should be chosen so that cells reach 50–70% confluency at the time of treatment. This density ensures there is enough space for the cells to grow without becoming overcrowded, which can confound results.
After seeding, cells are incubated overnight in a standard incubator at 37°C with a 5% CO2 atmosphere. This incubation period allows the cells to adhere firmly to the plate and recover from being transferred. Healthy, well-adhered cells provide a consistent baseline for the experiment.
Step-by-Step Cell Treatment
Prepare the final working concentrations of staurosporine by diluting the concentrated DMSO stock solution into fresh, pre-warmed cell culture medium. Add the staurosporine stock directly to the medium and mix it thoroughly to ensure a uniform concentration. This also prevents localized toxicity from the DMSO solvent.
A typical starting concentration for inducing apoptosis with staurosporine ranges from 0.5 to 2 micromolar (µM). The incubation time commonly falls between 3 to 6 hours, although some cell types may require longer exposure. These values are general starting points, as optimal conditions can vary significantly between different cell lines.
With the treatment medium prepared, carefully remove the existing culture medium from the cells by aspiration. Take care not to disturb the layer of adhered cells at the bottom of the well. The medium containing the desired concentration of staurosporine is then gently added.
The cells are then returned to the incubator under standard culture conditions, typically 37°C and 5% CO2. The duration of this incubation is a parameter of the experiment. It should be based on established literature for the specific cell type or determined through preliminary optimization experiments.
Methods for Detecting Apoptosis
A widely used technique for quantifying apoptosis is flow cytometry with Annexin V and propidium iodide (PI) staining. In early apoptosis, a lipid called phosphatidylserine flips to the outer surface of the cell membrane. Annexin V is a protein that binds to phosphatidylserine and, when labeled with a fluorescent dye, can identify these early apoptotic cells. PI is a fluorescent dye that enters late-stage apoptotic and necrotic cells where the membrane has lost integrity, allowing it to stain the DNA.
Another method for confirming apoptosis is Western blotting to detect the activation of executioner caspases. Caspase-3 is a primary executioner caspase that exists as an inactive pro-enzyme in healthy cells. During apoptosis, initiator caspases cleave this pro-enzyme into smaller, active subunits. A Western blot using an antibody specific to the cleaved form of caspase-3 will show a distinct band, confirming the caspase cascade has been activated.
Direct observation of morphological changes provides visual confirmation of apoptosis. Using a microscope, researchers can identify the characteristic physical signs of cells undergoing programmed cell death. These changes include the cell shrinking and detaching from the plate, membrane blebbing, and chromatin condensation. This condensation can be visualized with DNA-binding dyes like DAPI or Hoechst.
Essential Controls and Optimization
A vehicle control is necessary to ensure the observed cell death is a direct result of the staurosporine treatment. This involves treating a set of cells with the solvent used to dissolve the staurosporine, typically DMSO. The vehicle control should contain the highest concentration of DMSO that any of the staurosporine-treated cells are exposed to. This step rules out the possibility that the solvent itself is causing toxicity.
An untreated control group is also standard. This group of cells receives no treatment and is incubated in fresh culture medium for the same duration as the treated groups. This baseline sample shows the normal state of the cells and helps account for any spontaneous cell death that may occur.
A dose-response experiment involves treating cells with a range of staurosporine concentrations to find the one that induces a significant level of apoptosis without causing excessive necrosis. A time-course experiment involves treating cells for different lengths of time to identify the point at which apoptosis is clearly detectable.