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e., alters the total amount of cell membrane surface area), a non-penetrating, bright orange-red fluorescent dye (Cell Membrane Stain; excitation/emission ~555 nm/~565 nm) that evenly coats the outer surface of cells was selected as a non-specific neurite outgrowth reporter. Both the cell body PRDX4 and any neurite projections are uniformly stained with this dye (Fig. ?2A2A). Because this relatively sticky dye can also coat plastic tissue-culture surfaces, we found that adding a background suppression reagent following removal of the stain minimizes this background fluorescence and thereby eliminates the need for additional wash steps (Fig. ?1B1B). The suitability of this dye-based approach to stain for neurite outgrowth was tested by setting up a time-course in which cryopreserved rat cortex neurons were plated Duvelisib price under neurite outgrowth-promoting culture conditions and then stained at several different time-points up to 14 days after initiating the culture (Fig. ?2B2B). As expected, increased cell membrane staining was observed over time, indicative of significant neurite outgrowth occurring within this culture. It is important, however, to point out that since the Cell Membrane Stain is not neuron-specific it will label any cell-type, which precludes its use for discriminating between neuronal and non-neuronal cell types in mixed culture settings. Nevertheless, this cell membrane staining approach is suitable for mono-cultures in which a single cell-type (e.g., PC-12 cells) or a highly-enriched neuronal cell population (e.g., Fig. ?2C2C, selleck chemicals llc which indicates that most of the cells in the rat cortex neuron cultures used in these experiments stained positive for the neuronal markers MAP2 and beta-III tubulin, but not for the glial marker GFAP) is interrogated for phenotypic changes relating to cell membrane content or morphological presentation such as, but certainly not limited to, neurite outgrowth. Since neurite outgrowth is closely linked to neuronal cell health, it would be ideal to measure both parameters from the same sample. Accordingly, the acetomethoxy derivate of calcein (Calcein AM), a well-established cell viability reagent [11], was selected for inclusion alongside the Cell Membrane Stain. This cell-permeable dye is converted by intracellular esterase activity from a non-fluorescent substrate to a green-fluorescent product (Cell Viability Indicator; excitation/emission ~495 nm/~515 nm, Fig. ?2A2A). Notably, application of the Cell Viability Indicator reagent to neuronal cells results in differential staining, with relatively bright staining of the cell body and dim labeling of the neurite extensions.
