Multiplexing cell viability assays.

Today, obtaining mechanistic insights into biological, toxicological, and pathological processes is of upmost importance. Researchers aim to obtain as many as possible data from one cell sample to understand the biological processes under study. Multiplexing, which is the ability to gather more than one set of data from the same sample, fulfills completely this objective. Obviously, multiplexing has several advantages compared to single plex experiments and probably the most important one is that data on various parameters at exactly the same time point on the same cells or group of cells can be obtained and consequently this may contribute to saving time and effort and a reduction of the costs.In this chapter, different endpoints were measured starting from two-seeded multiwell plates, namely, cell viability, caspase-3/7 activity, lactate dehydrogenase (LDH), adenosine triphosphate (ATP), aspartate aminotransferase (AST), and glutamate dehydrogenase (GLDH) measurements. These -different endpoints were analyzed together to determine the cytotoxic properties of pharmaceutical compounds and/or reference compounds. A 96-well plate was designed to allow appropriate measurement of five doses of a compound in triplicate to determine the effect of the compound on the six different endpoints. The first four endpoints (cell viability, caspase-3/7 activity, LDH, and ATP) are discussed in detail in this chapter. AST and GLDH measurements are not discussed in detail as these are fully automatic measurements and thus behind the scope of this chapter.As an illustrating example, the reference compound tamoxifen was used to evaluate its cytotoxic properties using the hepatocellular carcinoma cell line HepG2 cells.