Alkaline phosphatase (ALP) is a ubiquitous membrane-bound glycoprotein that catalyzes the hydrolysis of phosphate monoesters at basic pH values. Examination of expression of ALP at intracellular and extracellular level is a widely-used procedure in both clinical practice and basic research. In the clinic, changes of normal ALP level in plasma is a routine diagnostic marker for various pathological processes (Sharma et al., 2014). At the basic research level, ALP quantification is frequently used in the study of cancer physiology and for evaluation of pluripotency in stem cells. Intracellular expression of ALP is very high in induced pluripotent (iPS) and embryonic stem cells (ES) and therefore can be used as unique and unambiguous biomarker of stem cells (Martins et al., 2014; Stefkova et al., 2015). In cancer, ALP expression inversely correlates with disease severity in advanced colon cancer and positively correlates with the ability of cells to differentiate (Shin et al., 2015).
Multiplexing is common in dual luciferase reporter assays, where transfection of Renilla and Firefly luciferase reporter plasmids are performed in batch and then read sequentially (Liu et al., 2009). A combination of two detection methods, such as luminescence and fluorescence, is also possible although frequently there is a loss of sensitivity as optimal conditions for one platform may not be compatible with the other and requires either expression of fluorescent biomarkers like Green Fluorescent Protein (GFP) or live staining of cells with a fluorescent dye, such as Calcein AM.
Materials and methods
In order to examine whether CDP could be effectively used as a reagent for detection of ALP in a multiplexed assay with metabolic proliferation reagents in-vitro, we prepared calf intestinal ALP into 1xCutSmart Reaction Buffer and then diluted in LB with serial dilutions to simulate conditions of cell lysis. Following CDP addition, we saw a linear luminescence signal in all range of concentrations: from 0.017nM to 20nM (data not shown). To further evaluate the compatibility of the ALP reaction with metabolic detection reagents, we added LB, CTG buffer, or ATPlite buffer. While addition of LB had no effect on the CDP signal, both CTG and PE buy GSK2656157 markedly quenched the CDP signal (Fig. 1, A). Notably, 6 other buffers used for cell viability detection were purchased from other companies (see Materials and methods) and had no effect on CDP signal (data not shown). Quenching of the CDP signal by subsequent addition of a viability reagent is useful as the secondary luminescent, reading from the viability reagent will not be convoluted by luminescence from two different enzymes in the same homogenous reaction.
CTG is a reagent used to determine the number of viable cells in culture based on quantification of the total ATP correlating with cell number. Since ATP and ALP are both presented in cell lysates, it was important to exclude the possibility that one of the agents impacts the luminescent signal of the other. We measured ALP activity between and 1000nM by CDP reagent in the presence (10, 100, or 1000nM) or absence of ATP, and observed no effect of ATP on ALP signal (Fig. 1, B). Conversely, there was no effect of ALP on ATP signal when measured in the presence of ALP in 7 different concentrations (0 to 241nM) using CTG (Fig. 1, C).
Elevated ALP expression is one of the ubiquitous markers of embryonic stem cells and pluripotent stem cells (Stefkova et al., 2015), and therefore a potential application for CDP/CTG methods is the determination of the cellular “stemness” in a complex culture of cells from multiple lineages. Different numbers of either induced pluripotent stem cells (iPSC) or mouse embryonic fibroblasts (MEF) cells were plated and CDP/CTG signals were measured at the next day. Signals from both CDP and CTG increased with cell number (Fig. 2, A, B) when examining iPS cells. In contrast, we observed almost no CDP signal in MEF cells, while CTG signal was correlated directly with cell number. Further, the sequential addition of the metabolic CTG reagent requires compatibility of the protocols and reagents in the same reaction well. Therefore, we compared different methods of sequential addition of CTG reagents following the CDP reaction. Cells were lysed by the CDP-compatible LB protocol and then either LB or CDP reagents were applied, followed by the addition of CTG. The original manufacturer\’s CTG protocol recommends aspiration of growth medium and direct addition to cells of CTG into remaining medium. When applied sequentially in cells, prior reactions of CDP did not adversely affect the CTG signal (Fig. 2, C), with similar results when CTG was applied directly without aspiration of media. Taken together the results suggest the ability of CTG usage after CDP in same well without affecting of experimental parameters.