Introduction
Apoptosis is a cellular program that enables superfluous or damaged cells to eliminate themselves almost without adverse effects on surrounding cells. This “programmed cell death” is a natural occurrence during development to give organs their shape, or to eliminate redundant or potentially harmful cells of the immune system in the adult organism. However, numerous findings indicate that apoptosis may be involved in many acute and chronic neurodegenerative diseases such as Parkinson, Huntington and Alzheimer diseases. Insufficient apoptosis can be found, for example, in cancer and autoimmune diseases. Hence, apoptosis and its effects are important factors in the investigation of diseases(1)
(2)
(3)
.
"Automating this [Apo-ONE® Homogeneous Caspase-3/7] assay eliminates errors resulting from pipetting inaccuracies, making available a standard protocol for reproducible performance of this assay."
The process of apoptosis can be divided into two phases: the initiation phase and the execution phase. The initiation phase can be triggered by intrinsic signals (i.e., the cell itself recognizes that it is superfluous or harmful) or by extrinsic signals, meaning that the cell receives external apoptotic signals. During apoptosis, caspases, enzymes with proteolytic activity, play a crucial role. In the initiation phase, the caspase cascade is triggered, which will activate the execution caspases, mainly caspases 3, 6 and 7(1)
(2)
(3)
. The activity of caspases 3 and 7 can be measured using the Apo-ONE® Homogeneous Caspase-3/7 Assay (4)
. Automating this assay eliminates errors resulting from pipetting inaccuracies, making available a standard protocol for reproducible performance of this assay.
Materials Required
- NIH/3T3 cells (mouse fibroblasts)
- DMEM
- 10% FBS superior
- trypsin 0.25% with EDTA
- DPBS
- Apo-ONE® Homogeneous Caspase-3/7 Assay (Cat.# G7790)
- staurosporine in DMSO
- micro-clear plate 96-well plates, black wells, flat bottom (Greiner Cat.# 655087)
- 15ml tubes
- plate reader (e.g., Tecan Safire2™ plate reader)
Table 1. Eppendorf Workstation and Required Labware List. 
Methods
NIH/3T3 mouse fibroblast cells cultivated in DMEM were seeded at a density of 33,000 cells/well onto a black 96-well multiwell plate with clear bottom (e.g., micro-clear plate, Griener Cat.# 655087). To ensure adhesion, the cells were incubated overnight at 37°C in a humidified 5% CO2 atmosphere.
The following day, the cells were treated with two different concentrations of staurosporine (1μM and 10μM), an apoptosis-triggering reagent. The staurosporine dilutions were prepared in medium in two 1.5ml Safe-Lock tubes and dispensed into each well of the 96-well plate using the Reagent Transfer method on the epMotion® 5075 TMX(5)
. The cells then were incubated for 5 hours at 37°C in a humidified 5% CO2 atmosphere. Following incubation, the Apo-ONE® Homogeneous Caspase-3/7 Buffer was transferred to a 15ml tube. Then the Apo‑ONE® Buffer and Substrate, along with the sample plate, were placed onto the worktable (5)
. The Apo-ONE® Reagent was mixed from the substrate and buffer, and 100μl of the Apo-ONE® Reagent were distributed into each well of the plate using the Reagent Transfer method on the epMotion® 5075 TMX (5)
. The plate then was incubated for 30 minutes at 37°C and shaken at 350rpm on the Thermomixer on the epMotion® 5075 TMX deck. At this point, the fluorescence could be measured using 499nmEx/521nmEm wavelengths in the Safire2™ plate reader. Medium with the Apo-ONE® Reagent was used as the blank value, and cells incubated without staurosporine but with Apo-ONE® Reagent were used as a negative control.
Results
Figure 2 shows that apoptosis of the NIH/3T3 cells intensifies with increasing concentrations of staurosporine. The fluorescence of the medium alone (blank) was subtracted from all other values. The average blank value was 853RFU. As expected, the autofluorescence of the cells in medium lay above this value. The maximum concentration of 10μM staurosporine was used to ensure cell death. The concentration of 1μM was chosen as a comparative value.
Figure 2. Staurosporine-induced apoptosis of NIH/3T3 cells. 
Conclusion
The Apo-ONE® Homogeneous Caspase-3/7 Assay is ideally suited for automation on the epMotion® 5075 TMX. The integrated Thermomixer allows assay incubation without further manual handling, thereby ensuring reproducible incubation conditions. In addition, the easy but flexible programming and use of the epMotion® 5075 TMX equipped with the epBlue™ user software allows for reproducible distribution of the appropriate dilutions of the cytotoxic substances.