A facile method for simultaneously measuring neuronal cell viability and neurite outgrowth.

Area of Science:

• Neuroscience
• Cell Biology
• Biotechnology

Background:

• Neurite outgrowth is crucial for neuronal function and health, but current measurement methods are time-consuming and labor-intensive.
• Existing techniques often rely on immunocytochemistry and microscopy, requiring manual or automated image analysis.
• There is a need for simpler, faster, and more quantitative methods to assess neurite outgrowth and neuronal health.

Purpose of the Study:

• To develop a rapid, simple, and multiplexed fluorescent dye-based assay for simultaneous measurement of neuronal cell health and neurite outgrowth.
• To provide an alternative to current laborious methods for assessing neuronal morphology and viability.

Main Methods:

• Developed a dual-color fluorescent staining method using an orange-red dye for cell membrane surface staining (indirectly indicating neurite outgrowth) and a green-fluorescent product from a cell-permeant dye for cell viability.
• The assay utilizes an orange-red fluorescent dye to report on changes in neurite outgrowth by staining cell membrane surfaces.
• Cell viability is assessed using a cell-permeant dye converted to a green fluorescent product by intracellular esterase activity.

Main Results:

• Successfully demonstrated the multiplex assay in Neuroscreen-1 cells, primary rat cortex neurons, and human induced pluripotent stem cell (iPSC)-derived neurons.
• The assay allows for rapid visualization and quantitative analysis of neuronal cell health and neurite outgrowth using a plate reader.
• Achieved unbiased, quantitative measurement of both neuronal health and relative neurite outgrowth from the same sample.

Conclusions:

• The developed dual-color fluorescent dye assay provides a quick, simple, and effective method for simultaneously assessing neuronal cell health and neurite outgrowth.
• This assay offers a significant improvement over traditional methods, enabling rapid, unbiased, and quantitative analysis.
• The method is versatile and applicable to various neuronal cell types, including iPSC-derived neurons, facilitating neuroscience research.