MCE CTG Cell Viability Detection Reagent (hereinafter referred to as CTG), the "gold standard" of cell viability detection. The homogeneous detection operation scheme of the CTG is "Add-Mix-Measure", Experience "Become visible" within 10 minutes.

Commonly used cell viability detection reagents mainly include MTT, CCK8, CTG and other detection methods (Fig. 1).

MTT : Also known as MTT colorimetric method. The detection principle is that succinate dehydrogenase in the mitochondria of living cells can reduce exogenous MTT to water-insoluble blue-purple crystal formazan and deposit in the cells, while dead cells have no such function. Dimethyl sulfoxide (DMSO) can dissolve formazan in cells, and its light absorption value is measured at a wavelength of 490 nm, which can indirectly reflect cell viability of living cells. Apart from the fact that the procedure is time consuming, the formed formazan is insoluble in water and can only be detected after being dissolved in DMSO. It increases the workload and cannot guarantee the accuracy of the measurement results, and, the solvent has obvious toxicity to human body.

CCK8 (Cell Counting Kit-8): Both the operation steps and detection time have been optimized a lot compared with MTT. CCK8 is a rapid, highly sensitive, non-radioactive colorimetric assay kit based on WST-8 and widely used in cell proliferation and cytotoxicity assays. But in experiments, there is a frequent question: "The more cells proliferate, the faster they grow, the darker will be the color of the suspension; In case of more cytotoxic cells, the lighter will be the color ", but I can't define the standard of this color.

CTG : It is used for detecting the number and viability of living cells in culture based on high-sensitivity bioluminescence detection technology of the adenosine triphosphate (ATP) present. This method has the advantages of "fast, accurate, and convenient", which is nearly 20 times faster than the processing speed of MTT, saving nearly 4 hours of time.

CTG's unique homogeneous detection method is "Add-Mix-Measure". Only need to add an equal volume of this reagent to the cultured cells for detection. So, why can't cell viability detection be so simple?

First, let’s comprehend the detection principle of the CTG luminescence method: As an important index of cell metabolism, ATP has a good linear relationship with the number of living cells. It is an important molecular marker for cell viability detection. The principle of ATP bioluminescence technology is as follows: luciferase uses luciferin, adenosine triphosphate (ATP) and O₂ as substrates, and converts chemical energy into light energy in the presence of Mg²⁺. In the luminescence reaction catalyzed by luciferase, the concentration of ATP is linearly related to luminescence intensity within a certain concentration range. The amount of ATP is directly proportional to the number of cells present. Based on this, the CTG Cell Viability Detection Reagent can be used for cell counting or viability determination by ATP content, and independent of compound autofluorescence (Fig. 2). It is ideal for cell proliferation assays, cell viability assays, and high-throughput screening assays for cytotoxicity.

Case 1: Cell Proliferation Assay

As shown in Fig. 3, in order to explore the effects of SIPL1 on the proliferation of TNBC cells, the SIPL1 expression vector was transfected into BT-549 and MDA-MB-231 cell lines. The cell proliferation rates were detected by CCK8 and CTG luminescent cell viability assay^[1].

CCK8 detection: TNBC cell lines were transiently transfected with shRNAs or overexpression plasmids and cultured for 24 h before detection.

CTG luminescent cell viability assay: TNBC cells were cultured at 37℃ overnight. Add 100 µL CTG luminescent cell viability assay to the culture medium, mix gently, incubated at room temperature for 20 minutes. The results demonstrated a marked decrease in cell proliferation in BT-549 and MDA-MB-231 cell lines transfected with shRNAs targeting SIPL1 (Fig. 3).

Case 2: Cell viability Assay

In order to evaluate the RRM2 suppresses ferroptosis in liver cancer cells, the exogenous RRM2 or two independent shRNAs targeting RRM2 were introduced into HepG2 and SMMC-7721 cells, and the cell viabilities were detected by CTG method (Fig. 4). The results demonstrated that RRM2 overexpression promoted cell viability, whereas RRM2 knockdown inhibited cell viability^[2].

Case 3: Cytotoxicity High Throughput Screening Assay

As show in the following figure, author teams report a phenotypic quantitative high-throughput screening (qHTS), based on concentration-response curves, which was designed to identify compounds active against Plasmodium sp., a liver and asexual blood stage parasite (Fig. 5). The qHTS used either SYBR Green or luciferase to test for inhibition of parasitic growth, assaying a combined total of 456,817 compounds.

Following qHTS, manual triage selected 4253 synthetically tractable compounds for validation of antiplasmodial activity and mammalian toxicity. Compounds were then evaluated in vitro against P. berghei liver stage parasites at 3 μM and 1 μM concentrations. Cellular toxicity against HepG2 was also assessed for all 4253 compounds using a CTG assay. 255 compounds exhibited some level of growth cellular toxicity against HepG2 cells, with AC50 values < 43 µM, and were subsequently excluded. Based on potency and chemical novelty, we ultimately selected 994 validated, non-toxic ABS hits for subsequent assessment against liver stage parasites^[3].

Aliquot the stock solution to routine usage volumes and store at -20℃.

1. Repeated freezing and thawing may cause a small amount of precipitation in the reagent. If residues remain after equilibration, they may be removed by centrifugation.
2. Please use a white or black multi-well plate (96-well plate or 384-well plate). Mutual interference may occur between adjacent wells of an ordinary transparent multi-well plate.
3. If the solvent content of the drug under test is high, the luciferase reaction and the light signal may be interfered. The control well of cell culture medium containing solvent can be set up to eliminate this interference.
4. This product is for R&D use only, not for drug, household, or other uses.
5. For your safety and health, please wear a lab coat and disposable gloves to conduct the work.