Cyto3D® Live-Dead Assay Kit

Cyto3D® Live-Dead Assay Kit Versatile live/dead assay kit for 3D & 2D Culture, Organoids, Spheroids, Stem Cells, Fluorescence Microscopy Versatile & Excellent for 3D/2D Cell Culture Works with cells cultured in hydrogels and animal-based ECM. Excellent for 3D cell culture. Fast & easy setup No pre-mixing required. Ready-to-use. Fast (5-15 minutes) one-step staining procedure for cell viability analysis. Even Staining & Clear Resolution Imaging No unevenness and no background staining for clearer imaging. The Cyto3D® Live-Dead Assay Kit is a versatile live/dead assay for 3D and 2D Cell Culture, Organoids, Spheroids, Stem Cells, and Fluorescence Microscopy. It is used to determine the live/dead nucleated cells using a quick one-step staining procedure for analysis on a dual-fluorescence system. The Cyto3D® Live-Dead Assay Kit is recommended for viability analysis of cells/organoids cultured in 3D, 2D coating, and on monolayer and works with cells cultured in animal-based ECMs and other hydrogel systems. Acridine orange (AO) and propidium iodide (PI), both nuclear staining (nucleic acid binding) dyes, are used in this kit. AO is permeable to 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 have 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 Specifications FormulationPremixed acridine orange (AO) and propidium iodide (PI), nuclear staining dyes  UseLive dead cell viability analysis for 3D and 2D cell culture Detection MethodFluorescent Excitation/Emission: AO (494/517nm), PI (535/617nm) Standard filtersAO (GFP), PI (Texas Red) For use with (Equipment): Fluorescence microscope, flow cytometer, microplate reader, fluorescence cell counter. Storage2 to 8°C (Protect from light) Shipping Conditions:Ships at ambient temperature Sizes1 mL Number of reactions500 (at 2 µL per 100 µL) Protocols and Resources Cyto3D® Live-Dead Assay Kit Protocol Product Documentation Sales Sheet – Cyto3D® Live-Dead Assay Kit Product Data Sheet Material Safety Data Sheet (MSDS) Frequently Asked Questions  Data and References Figure 1: Live-dead cell viability images: Intestinal organoids stained with Cyto3D® Live-Dead Assay Kit. Intestinal organoids were cultured in regulated conditions for 5 days. Six microliters of Cyto3D® reagent were mixed with organoid culture media (each well includes 150 µL of organoid culture media and 150 µL of hydrogel volume). The mixture was incubated at 37°C for 10-15 min, and the cells were observed under a fluorescence microscope. (A) A bright field image of a mature intestinal organoid. Images show live cells (B: Green) and dead cells (C: Red) in a mature intestinal organoid. Figure 2: Live-dead cell viability images: Intestinal organoids stained with Cyto3D® Live-Dead Assay Kit. Intestinal organoids were cultured in regulated conditions for 2-3 days. Six microliters of Cyto3D® Live-Dead Assay reagent were mixed with organoid culture media (each well includes 150 µL of organoid culture media and 150 µL of hydrogel volume). The mixture was incubated at 37°C for 10-15 min, and the cells were observed under a fluorescence microscope. (A) A brightfield image of a young, healthy intestinal organoid. Images show live cells (B: Green) and dead cells (C: Red) in a healthy intestinal organoid. Figure 3. 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. Two microliters (2 µL) of Cyto3D® Live-Dead Assay 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 fluorescence 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 using the Cyto3D® Live-Dead Assay Kit. Figure 4. Live-dead cell viability images of stem cell spheroids. Stem cells were static suspension-cultured in VitroGel® STEM (CAT# VHM02) for 5 days. Two microliters (2 µL) of Cyto3D® Live-Dead Assay 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 fluorescence 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. Resources using Cyto3D® Live-Dead Assay Kit White Papers A Quick Organoid Viability Measurement by Using Cyto3D® Live-Dead Assay Kit Webinars A Quick Organoid Viability Measurement by Using Cyto3D® Live-Dead Assay Kit Other Webinars Patient-Derived Tumoroids in 3D Xeno-Free Hydrogel with Automated Biomarker Detection Development of In Vitro Intestinal Model With Macrovascular Endothelium: Compare Animal-based and Xeno-free ECM Platforms Research Highlights | Application Notes Research Highlights Revolutionizing Cancer Research: A Patient-Derived Platform to Model Tumor-Immune Interactions Research Highlights Advancing Borderline Ovarian Tumor Research: Precision Viability Assessment in Patient-Derived Organoid Models Research Highlights Cyto3D® Live-Dead Assay Kit Aids in Identifying MiRNAs as Promising Biomarkers for Glioma Prognosis Research Highlights Check the MAP(K): Cancer Driver Signals on Pathway to Change Lanes Research Highlights The Good Side of Bad Cholesterol Application Notes 3D Spheroid Invasion Assay With the Xeno-free, Bio-Functional VitroGel® Hydrogel Matrix Application Notes Long Term 3D Tumor Spheroid Culture in VitroGel® Hydrogel Matrix Application Notes Advanced 3D Luminal Breast Cancer Model in VitroGel® System: Long-term 3D Cell Culture and Co-culture with Fibroblast Cells Application Notes Advanced Skin Cell Models Using the VitroGel® System: 2D Coating and 3D Cell Culture with Human Dermal Fibroblasts Application Notes New hPSCs Expansion Method – Direct from Liquid Nitrogen to 3D Culture Using a Xeno-free Hydrogel – VitroGel® STEM Application Notes Automated Dispensing, Monitoring, and Assay Development of Hydrogel-based 3D Cellular Models Application Notes Functionalizing Xeno-Free VitroINK® Bioinks to Promote Cell-Cell Interactions Between Human-Induced Pluripotent Stem Cell-Derived Testis Cells for In Vitro Spermatogenesis References/Publications Sorrentino, C., Lauretta, C., D’Angiolo, R., Musella, S., Giovannelli, P., Bertamino, A., Ostacolo, C., Gomez Monterrey, I., Migliaccio, A., Castoria, G., & Di Donato, M. 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EMBO Molecular Medicine, 13(10). https://doi.org/10.15252/emmm.202114123 View More Publications on VitroGel® Products