Green (calcein) stains for live cells, red (EthD) for dead cells. Microscope image of Live/Dead assay of neural stem cells. To follow along, you can download an example Live/Dead image here. It is available for Mac, Windows, and Linux platforms. While this approach can be used for 3D cell culture, the results are more reliable if the sample is relatively thin and there are not many cells overlapping in different focal planes.įor this analysis, we will be using free image processing software Fiji, an extended version of ImageJ. This workflow is suitable for images of 2D and thin 3D controls captured using a fluorescent or confocal microscope for samples stained with calceinAM/ethidium homodimer (EthD). This guide will walk you through a step-by-step guide on how to perform a Live/Dead quantification using Fiji. You can read more about different types of viability assays on our assays for 3D culture page. Dead cells are labeled with the ethidium homodimer dye (EthD) which binds to their DNA and fluoresces red. Live cells are stained with calcein and generate green fluorescence upon the excitation of their cytoplasm. Live/Dead assay is a very common cell staining procedure.
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