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AlphaLISA Caspase Assay and Apoptosis

Overview


Apoptosis or programmed cell death can be induced via two main pathways: the death receptor-mediated and the mitochondrial pathways. Caspases belong to the intracellular cytokine proteases class, which is central for apoptotic responses. By breaking down key cellular components that are required for maintaining normal cellular functions, caspases are responsible for executing morphological and biochemical consequences directly or indirectly attributed to apoptosis. Inhibitors, as well as inducers, of apoptosis might be suitable medicines for a variety of disease states including Alzheimer’s disease and cancer.

Human caspase-3, also known as apopain, is a cysteine protease of 29 kDa. It is widely expressed and is an integral component of the apoptosis cascade. Caspase-3 is synthesized as an inactive pro-enzyme that is processed in cells undergoing apoptosis by self proteolysis and/or cleavage by other upstream proteases. Its cleavage generates p17 and p12 subunits. The p17/p12 heterodimer associates with itself to form a heterotetramer. Once active, caspase-3 cleaves key cellular proteins such as poly(ADP-ribose) polymerase, sterol regulatory element binding proteins, and other caspase members. Activation of caspase-3 is commonly used as a biomarker for apoptosis assessment.

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Measuring human active caspase-3


The AlphaLISA® active caspase-3 kit (cat. no. AL278) was designed to quantify human active caspase-3 in buffered solution or in cell lysates using homogeneous (no wash steps) AlphaLISA technology. The kit uses a monoclonal antibody (clone 9H19L2) that is specific for the cleaved (active) form of caspase-3, while the second antibody is specific to the full length protein.

AlphaLISAcaspase 
Figure 1. Detection of caspase-3 in a sandwich antibody AlphaLISA assay format. In the AlphaLISA assay, biotinylated anti-caspase-3 antibody binds to the streptavidin-coated Donor beads while another anti-caspase-3 antibody is conjugated to AlphaLISA Acceptor beads. In the presence of the analyte, the beads come into close proximity. The excitation of the Donor beads provokes the release of singlet oxygen molecules that triggers a cascade of energy transfer in the Acceptor beads, resulting in a sharp peak of light emission at 615 nm.

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What do I need to run this assay?


Required reagents available from PerkinElmer:

  • Human active caspase-3 kit, which includes AlphaLISA Acceptor beads coated with an anti-caspase-3 antibody, streptavidin-coated Donor beads, biotinylated anti-caspase-3 antibody, lyophilized caspase-3 analyte, 5X AlphaLISA Lysis Buffer and 10X AlphaLISA Immunoassay Buffer. (Cat. No. AL278)
  • Microplates for cell culture/assay. We recommend our 384-well white CulturPlates™. Also see Microplate selection.
  • TopSeal™-A Adhesive plate seal for incubations

Available from other suppliers:

  • Protease inhibitor cocktail
  • Appropriate cells and cell culture medium
  • Orbital Shaker

Instrumentation/equipment:

  • A plate reader capable of reading Alpha assays (see Instrument options on the Alpha main page)

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Alpha products and catalog numbers


Complete listing of Alpha products and catalog numbers.

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Assay development


To improve the assay performance, several parameters need to be optimized:

  1. Number of cells per well
  2. Inducer concentration
  3. Incubation time for cell induction

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General protocol


Human active capase-3 can be detected in adherent or suspension cells. For the validation, HeLa (adherent) and Jurkat (suspension) cells were used. The following table summarizes the protocol used for each type of cells:

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Table 1. Protocol for a cell-based AlphaLISA caspase-3 assay

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Data


Cell titration

Optimal number of cells were determined by testing increasing number of Jurkat and HeLa cells (from 2,500 to 30,000 cells per well), cultured in RPMI 1640 or MEM/EBSS medium supplemented with 10% FBS, respectively. Cells were stimulated with 30 mM staurosporine (STS) prepared in MEM/EBSS (FBS-free) media and incubated at 37°C (5% CO2) for 4 hours. Cells were then lysed with 1.5X AlphaLISA Lysis Buffer. Non-stimulated cells were used as controls. For both cell lines, the maximum signal to background ratio value (S/B) was obtained with 15,000 cells/well (Figure 2).

The S/B was calculated using the maximal signal (staurosporine-stimulated cells) and the minimal signal (non-stimulated cells).

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Figure 2. Cells titration experiment was performed using adherent (left panel) or suspension cells (right panel).

A cell titration experiment, using Jurkat cells, was also performed using a plate containing lysed cells frozen overnight, in parallel to a second plate where cells were lysed immediately prior to the caspase-3 AlphaLISA assay. The results are summarized in Table 2.

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Table 2. Jurkat cell titration using fresh cell lysates compared to a cell lysate preparation that was frozen overnight prior to use.

This experiment showed that an optimal assay window is obtained with 10,000 - 20,000 cells/well, but a good S/B ratio value could be generated at low cell density (2,500 cells/well). Moreover, similar results were obtained using both fresh and previously frozen lysates.


Time course

Time course experiments for each incubation period were then carried out. Results are presented in Figure 3. After a 2-hour incubation, S/B values of 5 and 7 were obtained with HeLa and Jurkat cells, respectively, compared to S/B values of 11 and 10 after 4 hours. An incubation of 24 hours was also evaluated but due to the small volume of reaction, well evaporation led to cell death. A final incubation time of 4 hours was chosen for further experiments, though the 2-hour incubation also showed great assay performance (Table 3).

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Figure 3. Detection time course experiment performed with adherent (HeLa cells, left panel) or suspension (Jurkat cells, right panel) cells.
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Table 3. Time course experiment performed with Jurkat cells.


Compound dose-response curves

Staurosporine (STS) dose-response curves were performed using different cell lines. Jurkat, HeLa and MCF-7 cells (15,000 cells/well) were treated with increasing STS concentrations, from 3x10-10 to 1x10-4 M, for 4 hours. Non-stimulated cells were used as controls. The adherent cell protocol was followed for assays performed with HeLa and MCF-7 cells. For Jurkat cells, the protocol for suspension cells was used (see Table 1).

The specificity of the cell-based caspase-3 AlphaLISA assay was investigated by taking advantage of the MCF-7 breast carcinoma cell line. MCF-7 cells have lost the caspase-3 protein due to a 47-base pair deletion within exon 3 of the CASP-3 gene. Therefore, this cell line was used as a negative control. As expected, active caspase-3 was not detected in MCF-7 cells. A great STS dose-dependent response was seen in both HeLa and Jurkat cells, with EC50 values of 2.77x10-6 and 2.18x10-6, respectively. The results are illustrated in Figure 4.


Staurosporine_response_curves_ASK.jpg
Figure 4. Staurosporine dose-response curves using three different cell lines.

Assay reproducibility

The intra-assay variability was evaluated by Z’ factor value. The assay was executed two separate times and was performed in three ways, using 48 wells per treatment, as follows:

  1. 15,000 cells/well treated with or without 30 µM staurosporine
  2. 15,000 cells/well treated with or without 1 µM staurosporine
  3. 2,500 cells/well treated with or without 30 µM staurosporine


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Table 4. Intra-assay variability

The inter-assay precision was determined from two independent experiments, performed as 48 replicates. The average values for maximum counts, minimum counts, and statistical parameters from the combined experiments (n=96) are given in Table 5.


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Table 5. Inter-assay precision

In summary, Table 6 shows optimal parameters for each of the two cell lines used to conduct development of the cell-based caspase-3 assay.

AlphaLISA_caspase-3_ HeLa_Jurkat_ASK.jpg
Table 6. Optimized conditions for the AlphaLISA caspase-3 assay using HeLa and Jurkat cell lines.

* RPMI contains biotin

** As the cells are attached and the medium is removed, the volume added is not relevant.

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Posters, guides and reference materials


  • AlphaLISA reference tables contain information related to species selectivity, lower and upper limits of detection, dynamic range, and a description of sample matrices tested for each AlphaLISA kit.

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Tips


  • For adherent and suspension cells, low passages should be maintained in full growth media without antibiotics.
  • Adherent cells should not be allowed to grow to confluence and suspension cells should be maintained in a logarithmic growth phase. Follow manufacturer instructions for cell line-specific passaging conditions and culture media recommendations. Useful guides can be found at the ATCC website.
  • For cell-based assays, it is highly recommended to use a medium free of biotin for the treatment of cells to avoid signal quench and/or loss of sensitivity.
  • Adherent cells should be seeded the day before performing the AlphaLISA assay to permit adequate adherence to the surface.
  • The addition of protease inhibitors to the AlphaLISA Lysis Buffer is highly recommended.
  • For quantitative analysis of active caspase-3 in cell lysates, sample concentrations can be interpolated from a calibration curve. It is highly recommended to use a non-stimulated cell lysate to dilute the standard for the calibration curve. Furthermore, spike & recovery experiments should be performed to confirm that the caspase-3 can be accurately quantified.

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