SYTOX* Green

AP-induced toxicity and measurement of cell survival/injury were performed as described in detail elsewhere.1011 Briefly, 6-day-old cells were exposed to fresh solutions of either Ap25 35 or Ap, 42 for 24 h, in the presence or absence of different drugs. Cell survival and extent of cell death were determined using MTT and Sytox green assays, respectively. Measurement of intracellular reactive oxygen species was determined by dichlorofluorescein (DCF) fluorescence assay, as described previously.23... 

Green, L.C., LeBlanc, P.J. and Didier, E.S. (2000) Discrimination between viable and dead encephalitozoon cuniculi (microsporidian) spores by dual staining with Sytox Green and Calcofluor White M2R. Journal of Clinical Microbiology 38, 3811-3814. 

Loss of cytoplasmic membrane function occurs early in necrosis and late in apoptosis (so-called apoptotic necrosis ). If, in a given experimental system, the mode of death has been adequately characterized morphologically and the assay timepoint judiciously chosen, loss of dye exclusion can serve as a useful, easy to quantify measure of cell death in either apoptotic or necrotic model systems. Commonly used dyes for this type of analysis include propidium iodide, 7-AAD, Sytox green, and ethidium homodimer. 

Figure 1. NETs formed in vitro (stained with Sytox Green, 600x, by K. Fritzsche). 

Fluorescent nuclear stains (optional) DAPl, SYTOX Green (Molecular Probes , Life Technologies , Carlsbad, CA), TO-PRO -3 (Life Technologies ). 

Fig. 4 3D reconstruction of the rat neocortical microgliocyte (cell body and processes) created from 15 pm z-stack using the confocal laser microscope LSM 710 (Zeiss) and the software ZEN 2012 Gray (Zeiss). Ibal immunocytochemistry, fluorescence visualization—Cy3, nuclear counterstaining—SYTOX Green... 

In most cases, it is convenient to use a conjugate of streptavidin with the fluorochrome Cy3 which has a red flnorescence and the SYTOX Green dye for nuclear staining, which has a green fluorescence. Typically, most flnorescence microscopes have filters for these two fluorochromes, and confocal microscopes are equipped with lasers for the excitation of these dyes. 

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