Luciferase-based Assays for Apoptosis and Cytotoxicity (High Throughput Screening)

 

          Various cell fates exist in the in vitro cell cultured cells, including decreased cell viability, apoptosis (programmed death), and necrosis (accident death). There are multiple techniques to determine apoptosis, cell viability and cytotoxic activity by various biochemical and cell-based assay methods, such as measuring intracellular adenosine triphosphate (ATP) levels, cell membrane integrity, lactate dehydrogenase (LDH) release, the chromium (51Cr) release assay, flow cytometry and high-content imaging methods. However, there is still urgent need to have a robust method that is rapid, scalable, and reproducible. Ideally, it is desirable to have a cell-based assay that can be performed in a homogeneous format, in which reagents are simply added, and signal is detected without the need to remove supernatant or to wash the assay plate. Specially, the fate of the certain cell population in co-culture system can be directly measured, without separating the certain cells from co-culture system. Here, we develop a feasible luciferase-based screening assay that is validated for precise and accurate in vitro assessment of compound molecule and antibody potency and efficacy, such as Antibody-dependent cell-mediated cytotoxicity (ADCC), and also has the capacity to evaluate multiple tumor cell lines.

 

Luciferase-based Cellular Cytotoxicity Assays (High Throughput Screening)

 

          This cell-based assay is to measure the intracellular luciferase activity. Briefly, a luciferase expressing stable target (tumor) cell line would be established with lentiviral transduction. When the luciferase-expressing target cells are challenged with small molecule compounds, antibodies, effector cells such as T cells, target cell’ viability will be determined via quantification of constitutively expressed intracellular luciferase. The readout is endpoint-driven (target cell lysis). Compared to the intracellular luciferase activity in the untreated target cells, specific cytotoxicity of compounds, antibodies, and effector cells on the target cells would be calculated. Two types of luciferase-based assay for cytotoxicity: Firefly and Renilla luciferases, were available.

 

Luciferase-based Apoptosis Detection (High Throughput Screening)

 

          Apoptosis, programmed cell death, is a normal activity of multicellular organisms. Insufficient or excess of apoptosis is usually observed in many human diseases such as cancers, autoimmune disorders, diabetes, Alzheimer's, organ and bone marrow transplant rejection etc. Current methods detecting apoptosis include fluorescent-based staining approaches, such as ethidium bromide and acridine orange (EB/AO), DAPI (4; 6-diamidino-2phenylidole), Hoechst staining, DNA ladder, TUNEL (Terminal deoxynucleotidyl transferase mediated dUTP Nick End Labeling), and enzyme-based approaches, such as, Caspase-3/7 activity. However, these methods are involved in multiple steps, expensive and time-consuming, and even false-positive. To overcome these shortcomings of current methods, we developed a high throughput method to quantify apoptosis of adherent cells in a 96-well plate format.

 

          Phosphatidylserine (PS) in living cells, are primarily restricted to the inner membrane leaflet. However, PS would be translocated from the inner leaflet of the plasma membrane to the outer leaflet during the early to middle stages of apoptosis. Therefore, its surface expression makes it a potential indicator for apoptosis. 

 

          Annexin V, one of family of the annexins, binds phosphatidylserine (PS) in a Ca2+-dependent manner. The commercially available dye-labeled Annexin V, is widely used to quantify apoptosis by FACS, as the standard protocol in cell biology research, due to its binding cell-surface PS with high specificity and affinity. However, it was difficult to do high throughput screening of apoptosis using dye-labeled annexin V FACS. We thus over-expressed Annexin V protein and luciferase as the fused protein (Annexin V-Luciferase, Annexin V-luc). The fused Annexin V-luciferase can be used to monitor apoptosis with high throughput manner. The luciferase signal gave a wider dynamic range than other methods. A workflow of high throughput screening of apoptosis using Annexin V-Luciferase was outlined below.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

     Fig.1 Cross-section from a well of a 96-well microplate illustrating the principle of the apoptosis assay (Cat#: AL100 or ExoQA). Adherent cells, induced apoptosis cells, Annexin V-Luc, substrate ad signal were indicated.