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Trypan Blue is one of the most commonly used dyes for assessing cell viability by dye exclusion assays. Dye exclusion assays are based on the principle that an intact cell membrane is necessary for the exclusion of certain dyes. Some other dyes used for this purpose include safranin, eosin, Congo Red, erythrocin, nigrosin and Alcian Blue (5). Pappenheimer (4) was the first to describe the use of Trypan Blue for analyzing cell viability. Since then, Trypan Blue viability staining has become one of the most common methods of determining loss of cell viability, particularly at the time of subculture, because the method is inexpensive, dependable and efficient. It is likely that other laboratories have also developed their own methodologies for applying Trypan Blue staining to adherent cells growing in monolayer culture, but this is the first report that actually describes a method allowing for fixation and permanent mounting of Trypan Bluestained cell cultures grown on coverslips. Allison and Ridolpho (1) described a method by which cells could be Trypan Blue-stained, fixed and permanently mounted on a slide, but they used cells grown in suspension and sedimented onto the slide rather than monolayer cultures grown on coverslips as reported here. If in situ Trypan Blue staining is done on monolayer cultures without fixation and permanent mounting, then the water-soluble stain will eventually leach out of the cells and disappear. However, once fixed and permanently mounted, the Trypan Blue stain is permanently fixed into the tissue and will not fade over time. This method, because it is done in situ on monolayer cultures and incorporates a permanent mounting step, leads to several key advantages over traditional in situ Trypan Blue techniques. First, this method provides the convenience of analysis at any later time. Second, it greatly facilitates the use of computerized morphometric analysis to quantify the data. Finally, it allows for the use of double-staining techniques in conjunction with Trypan Blue, and we describe one such doublestain method subsequent to this report. This method has been extremely useful in our laboratory’s cytotoxicity studies using both primary human neuronal cultures (that are heterogeneous in composition) and neuronal cell lines. The technique described here can be applied to any cell line, because Trypan Blue staining is known to be widely applicable to all cell types. The protocol is provided in Table 1. Regarding step 4, note that many different Trypan Blue concentrations are seen in the literature, ranging from 0.04%–0.4%. The concentration can be varied within this range to achieve the desired staining intensity. Low concentrations around 0.04% are typically used in instances where the cells have been trypsinized, and it is likely that they will be exposed to the Trypan Blue for more than a few minutes, such as for cells in suspension at the time of subculture. Lower concentrations are necessary in these circumstances because the action of the trypsin alters the membrane of viable cells so that they will gradually take up Trypan Blue (6). However, because the membranes of cells in monolayer culture have not been altered by trypsinization, higher concentrations of Trypan Blue can be safely applied without compromising the integrity of viable membranes, provided that staining times are kept at 1 min or less. At a concentration of 0.2%, there is a slight variation in staining intensity among cell types, but this concentration works well for our primary neurons, glial cells and neuronal cell lines. Regarding step 5, other fixatives that we have tried that have no detrimental effects on the Trypan Blue staining include 2.5% glutaraldehyde for 30 min at 20°–22°C and 4% PFA/1% glutaraldehyde for 10 min at 20°–22°C or 20 min at 0°C. Post-fixes in 66% ethanol/33% glacial acetic acid for 5 min at -20°C also do not decrease the intensity of staining. In summary, we have detailed a protocol for fixing and mounting coverslips of adherent cell cultures that have

In Situ Trypan Blue Staining of Monolayer Cell Cultures for Permanent Fixation and Mounting