Cell Viability Assay

Cell viability refers to the number of live and healthy cells in a sample. Cell viability assay is widely used for screening small molecules, which are the largest class of therapeutics targeting neurological diseases.

In addition to providing reliable in vitro models of neurological disorders, Creative Biolabs is capable of offering a range of cell viability assays based either on reduced neuronal activity, metabolic activity, cellular ATP production, or live cell imaging in real-time to support your screening for any compounds of interest.

Real-Time Viability Assay

Unlike endpoint approaches, real-time assay provides a way to monitor viability throughout the time course of an experiment. This is particularly useful for assessing the impact of drugs on non-dividing cells, such as neurons. By adding non-perturbing cytotoxicity dye, we can positively mark and quantify living and dead cells. Moreover, we can obtain multiple cytotoxicity readouts using a combination of live cell nuclear stain, Caspase 3/7 dye, or Annexin V dye to measure proliferation and apoptosis alongside cytotoxicity.

Fig. 1 Real-time detection of apoptotic cells with both-Q-annexin V and PI.¹

Visualization of Cell Death

Once cell death is induced, caspase activation, cell surface exposure of phosphatidylserine (PS), the loss of membrane integrity, and DNA fragmentation will occur. As the most intuitive means, confocal imaging can observe these changes through the use of dyes, such as propidium iodide (PI), Caspase 3/7, Annexin V, cytochrome c. The detection can be done on classical dissociated in vitro cultures (e.g. primary neurons, microglia, astrocytes) but also on ex vivo brain slices. Meanwhile, super-resolution imaging offers nanoscale insights into cellular processes and drives advances in drug screening.

Fig. 2 Confocal imaging to detect apoptotic Annexin V translocation in neurons and astrocytes treated with ischemic solution (IS).²

MTT Assay

MTT is a positively charged, soluble and cell permeable tetrazolium compound. MTT assay is a commonly used colorimetric assay to measure cell viability and its principle is that succinate dehydrogenase in mitochondria in living cells reduces the water-soluble yellow tetrazolium salt MTT to water-insoluble purple formazan crystals. After adding a solubilizing reagent such as DMSO, record the absorbance at 570 nm to quantify the precipitate of dissolved formazan crystals, which can indirectly reflect the cell viability of living cells.

Fig. 3 Alzheimer's disease (AD) astrocytic-origin extracellular vesicles (AEVs) and neuronal-origin extracellular vesicles (NEVs) impair neuronal viability, which was measured by MTT assay.³

MTS Assay

MTS is a negatively charged tetrazolium compound and does not readily penetrate cells, similar to XTT, WST-1. MTS assay is more convenient than MTT assay because MTS combined with intermediate electron coupling reagents is reduced by living cells to produce formazan that is soluble in the cell culture medium. The level of formazan produced is measured by absorbance at 460 nm and is directly proportional to the number of living cells.

Fig. 4 MTS assay to detect the viability of the neural progenitor cells after treatment with MP and the EDNRB agonist.⁴

CCK8 Assay

CCK8 assay is based on dehydrogenase activity detection in viable cells, and the produced yellow-color formazan is water soluble and measured by absorbance at 450 nm. Compared to colorimetric assays using other tetrazolium salts such as MTT, XTT, MTS, or WST-1, CCK8 assay demonstrates significant improvements in detection sensitivity, operational efficiency, and detection speed.

Fig. 5 Cell viability was measured by CCK-8 (A, B). The results show that pretreatment with RSPO3 has a neuroprotective effect on Neuro-2a neuronal cells (A), the primary murine cortical neurons (B).⁵

Resazurin Assay

Resazurin assay aims at cell viability by fluorescent method and is based on the same principle as tetrazolium assays. After being reduced by living cells, blue resazurin changes to pink fluorescent resorufin, which can be measured by absorbance at 570 nm. Resazurin assay is a cost-effective and highly sensitive method compared to tetrazolium assays.

Fig. 6 Combined exposure to METH and Poly (I:C) results in significant neuronal damage in mixed-glial co-cultures.⁶

LDH Assay

LDH assay is a well-established assay for cell viability. LDH (lactate dehydrogenase) is a stable cytoplasmic enzyme present in many cells, so its detection indicates that the cell's membrane has been damaged or disrupted, suggesting the cell is either undergoing programmed cell death (apoptotic) or experiencing cell death due to injury or stress (necrotic). LDH assay is based on an enzymatic coupling reaction. The released LDH amount is directly proportional to the number of dead or damaged cells.

Fig. 7 LDH release assay was used to assess cytotoxicity levels of memantine, parbendazole, and bortezomib at varying doses on Day 2.⁷

ATP Assay

ATP assay is widely used for determining the number of live cells in culture based on quantitation of ATP present, a marker of viable cells. Colorimetric, fluorescent and bioluminescent methods can be used to perform ATP assay. Among them, bioluminescent ATP assay takes advantage of the firefly luciferase enzymatic reaction, and has the advantages of more sensitive, homogeneous, fast, accurate, and convenient.

Fig. 8 Effect of mitochondrial inhibitors on the survival of cultured astrocytes. (A) Cell viability by trypan blue. (B) Cell viability by ATP assay. (C) Cell viability by MTT assay.⁸

Creative Biolabs is passionate about providing you with a series of services for phenotypical and functional characterization of your compounds of interest with our cutting-edge platform and professional technical staff. If you are interested in our cell viability or related assays, please feel free to reach out to us for more information.

References

1. Kim, Hyunjin et al. "A Quenched Annexin V-Fluorophore for the Real-Time Fluorescence Imaging of Apoptotic Processes In Vitro and In Vivo." Adv Sci (Weinh). 2020, 7(24):2002988.
2. Pamenter, Matthew E et al. "Autophagy and apoptosis are differentially induced in neurons and astrocytes treated with an in vitro mimic of the ischemic penumbra." PloS One. 2012, 7(12):e51469.
3. Nogueras-Ortiz, Carlos J et al. "Astrocyte- and Neuron-Derived Extracellular Vesicles from Alzheimer's Disease Patients Effect Complement-Mediated Neurotoxicity." Cells. 2020, 9(7):1618.
4. Li, Shiyuan et al. "EDNRB Reverses Methylprednisolone-Mediated Decrease in Neural Progenitor Cell Viability via Regulating PI3K/Akt Pathway and lncRNA Expression." Journal of molecular neuroscience. 2020, 70(3):403-412.
5. Liu, Ting-Tao et al. "Endothelial cell-derived RSPO3 activates Gαi1/3-Erk signaling and protects neurons from ischemia/reperfusion injury." Cell death & disease. 2023, 14(10):654.
6. Alvarez-Carbonell, David et al. "Cross-talk between microglia and neurons regulates HIV latency." PLoS pathogensl. 2019, 15(12):e1008249.
7. Bayraktar, Abdulahad et al. "Drug repositioning targeting glutaminase reveals drug candidates for the treatment of Alzheimer's disease patients." Journal of translational medicine. 2023, 21(1):332.
8. Silva, Ellen A et al. "Proliferating Astrocytes in Primary Culture Do Not Depend upon Mitochondrial Respiratory Complex I Activity or Oxidative Phosphorylation." Cells. 2023, 12(5):683.