For single files like this, simply go to File > Open and select your file. tif image exported from the microscope software. There are instructions for modifications included directly in the macro file. You can follow the steps below for the manual quantification, but you can also download our Fiji macro and install it by going to Plugins > Macros > Install… and then use it by selecting it from Plugins > Macros > “live dead quantification.” You might need to modify specific settings of the macro to suit your needs by going to Plugins > Macros > Edit and selecting our macro file. 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. Furthermore, the ratio of ONL area to total retinal area showed a reduction in retinal area for the detached retinal sections and the ratio of ONL to total retinal thickness showed that detached retina had significantly reduced ONL thickness.Īutomated analysis using an image analysis program available publicly from the NIH is accurate and may be used in place of manual evaluation to reduce the time needed for data collection considerably.Live/Dead assay is a very common cell staining procedure. The cell area was used to obtain a more accurate value for photoreceptor nuclei count-this was accomplished by first determining the average cell area and a standard deviation which were used to construct a confidence interval to eliminate any outliers. Average cell area was calculated and plotted. Two other measurements were also assessed: the ratio of ONL area to total retinal section area and the ratio of ONL thickness to total retinal thickness.ĭetached retinal sections had significantly lower photoreceptor nuclei counts than attached retinal sections and automated cell counting produced accurate results. The number of nuclei in the ONL was determined with ImageJ and compared to manual counting. Parameters in ImageJ were adjusted to improve the accuracy of automated counting. Mean size of photoreceptor nuclei in attached and detached samples were compared. Automated cell counting was performed in the outer nuclear layer (ONL) of retinal sections where photoreceptor nuclei are located. Sections were examined using ImageJ, a Java-based image-processing program available by the National Institutes of Health (NIH). Retinal sections were stained and photographed. Eyes were obtained 1 and 2 months post-retinal detachment and processed for histologic analysis. To develop an automated, accurate way of analyzing different parameters of retinal sections to determine the effects of retinal detachment.Įxperimental retinal detachments were created in adult C57/B16 mice.
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