Cyto3D® Live-Dead Assay Kit

The Cyto3D® Live-Dead Assay Kit is used to determine the live/dead nucleated cells by using a fast one-step staining procedure for analysis on a dual-fluorescence system. This kit is recommended for viability analysis of cells/organoids cultured in 3D, 2D coating, and on monolayer.

• Ready-to-use

• Fast

• Sensitive

• Excellent for 3D cell cultures

• Cost-effective

Acridine orange (AO) and propidium iodide (PI), both nuclear staining (nucleic acid binding) dyes, are used in this kit. AO is permeable to both live and dead cells and stains all nucleated cells to generate green fluorescence. PI only penetrates the membranes of nucleated cells with compromised membranes and stains the dead cells to generate red fluorescence. Due to the quenching, when cells are stained with both AO and PI, all live nucleated cells fluoresce green and all dead nucleated cells fluoresce red (the PI reduces the fluorescence intensity of the AO by fluorescence resonance energy transfer (FRET)). Non-nucleated materials such as red blood cells, platelets and debris do not fluorescence and are ignored by fluorescence microscopes.

Dual-Fluorescence Viability, using AO and PI, is the recommended viability analysis method for cell lines, primary cells, and stem cells.

Easy setup and use

DATA AND REFERENCES

Figure 1. Live-dead cell viability analysis by using Cyto3D Live-Dead Assay Kit.
Glioblastoma cells (SF 298, about 60% cell viability) were 3D cultured in VitroGel system for 2 days. 2 µL of Cyto3D reagent was added to each well containing 50 µL hydrogel and 50 µL cover medium. The mixture was incubated at 37 °C for 5-10 min. The cells were then observed under a fluorescent microscope. The images show the Live (green) and Dead (orange) cells within the 3D hydrogel matrix. The z-stack images of cells within hydrogel were then 3D reconstructed and shown in the 4D view images. The live and dead cells at higher levels of the hydrogel are clearly shown in the images by using Cyto3D Live-Dead Assay Kit.

Figure 2. Live-dead cell viability images of stem cell spheroids.
Stem cells were static suspension-cultured in VitroGel STEM (CAT# VHM02) for 5 days. 2 µL of Cyto3D reagent was added to each well containing 100 µL cell suspension. The mixture was incubated at 37 °C for 5-10 min. The cells were then observed under a fluorescent microscope. The images show the Live (green) and Dead (orange) stem cell spheroids cultured in a 3D hydrogel matrix. The live-dead dyes of Cyto3D Live-Dead Assay Kit can successfully penetrate into large cell spheroids for cell viability analysis.

References/Publications

• Wang, H., Zhang, Y., Miao, H., Xu, T., Nie, X., & Cheng, W. (2024). CircRAD23B promotes proliferation and carboplatin resistance in ovarian cancer cell lines and organoids. Cancer Cell International, 24(1). https://doi.org/10.1186/s12935-024-03228-1

• Miao, H., Meng, H., Zhang, Y., Chen, T., Zhang, L., & Cheng, W. (2024). FSP1 inhibition enhances olaparib sensitivity in BRCA-proficient ovarian cancer patients via a nonferroptosis mechanism. Cell Death & Differentiation, 1–14. https://doi.org/10.1038/s41418-024-01263-z

• Babl, N., Hofbauer, J., Matos, C., Voll, F., Ayse Nur Menevse, Rechenmacher, M., Mair, R., Philipp Beckhove, Herr, W., Siska, P. J., Renner, K., Kreutz, M., & Schnell, A. (2023). Low-density lipoprotein balances T cell metabolism and enhances response to anti-PD-1 blockade in a HCT116 spheroid model. Frontiers in Oncology, 13. https://doi.org/10.3389/fonc.2023.1107484

• Belén, A., Sacconi, A., Tremante, E., Lulli, V., Caprara, V., Rosanò, L., Goeman, F., Carosi, M., Marta Di Giuliani, Vari, G., Silvani, A., Pollo, B., Garufi, C., Ramponi, S., Simonetti, G., Ciusani, E., Chiara Mandoj, Stefano Scabini, Villani, V., & Agnese Pò. (2023). A diagnostic circulating miRNA signature as orchestrator of cell invasion via TKS4/TKS5/EFHD2 modulation in human gliomas. Journal of Experimental & Clinical Cancer Research, 42(1). https://doi.org/10.1186/s13046-023-02639-8

• Wan, Y., Zhang, Y., Meng, H., Miao, H., Jiang, Y., Zhang, L., & Cheng, W. (2023). Bractoppin, a BRCA1 carboxy-terminal domain (BRCT) inhibitor, suppresses tumor progression in ovarian borderline tumor organoids. Biochemical and Biophysical Research Communications, 638, 76–83. https://doi.org/10.1016/j.bbrc.2022.11.063

• Di Donato, M., Galasso, G., Giovannelli, P., Sinisi, A. A., Migliaccio, A., & Castoria, G. (2021). Targeting the Nerve Growth Factor Signaling Impairs the Proliferative and Migratory Phenotype of Triple-Negative Breast Cancer Cells.Frontiers in Cell and Developmental Biology, 9. https://doi.org/10.3389/fcell.2021.676568

• Jiang, Y., Li, S., Zhou, Q., Liu, S., Liu, X., Xiao, J., Jiang, W., Xu, Y., Kong, D., Wang, F., Wei, F., & Zheng, C. (2021). PDCD4 Negatively Regulated Osteogenic Differentiation and Bone Defect Repair of Mesenchymal Stem Cells Through GSK-3β/β-Catenin Pathway.Stem Cells and Development. https://doi.org/10.1089/scd.2021.0041

• Uhlitz, F., Bischoff, P., Peidli, S., Sieber, A., Trinks, A., Lüthen, M., Obermayer, B., Blanc, E., Ruchiy, Y., Sell, T., Mamlouk, S., Arsie, R., Wei, T., Klotz‐Noack, K., Schwarz, R. F., Sawitzki, B., Kamphues, C., Beule, D., Landthaler, M., & Sers, C. (2021). Mitogen‐activated protein kinase activity drives cell trajectories in colorectal cancer. EMBO Molecular Medicine, 13(10). https://doi.org/10.15252/emmm.202114123