Measuring Neuronal Cell Health through Viability and Neurite Outgrowth

Tools that measure the fitness of neural cells are essential for monitoring the effects of a variety of factors, including biological modifiers, neural cell culture conditions, drug compounds, and environmental neurotoxicants. In particular, neural cell viability and neurite outgrowth are two of the most commonly measured indications of neural cell health and function. For example, researchers investigating novel therapeutic strategies to address neurodegenerative disorders typically need to determine whether their investigative approach improves neural function while minimizing any cytotoxicity-associated effects. Ideally, both parameters, neural cell viability and neurite outgrowth, are measured in the same sample to distinguish whether one or both aspects of neural cell fitness are affected.

The Molecular Probes® Neurite Outgrowth Staining Kit provides a two-color fluorescence stain that allows for simple, rapid visualization and quantification of neurite outgrowth and cell viability in the same sample. Neurite outgrowth is monitored via bright orange staining (excitation/emission ~555 nm/~565 nm) of outer cell membrane surfaces. Cell viability is simultaneously measured via the use of a cell-permeable viability indicator dye that is converted by living cells to emit green fluorescence (excitation/emission ~495 nm/~515 nm). A typical workflow using this kit requires 15 – 30 minutes and a few simple steps prior to taking a measurement.

Live cell and fixed cell protocols are provided below that are suitable for imaging/fluorescence microscopy or microplate reader analysis.

Important: The Neurite Outgrowth Staining Kit is not recommended for: discriminating between neuronal and non-neuronal cell types (i.e., the dyes used in this kit are not specific for neuronal cells, but will stain all cell types); use with cell permeabilization protocols; real-time imaging applications.

Required Materials

Cells

•  Neural Cells

Note: For comparing relative neurite outgrowth, it is recommended that appropriate controls are included in the experiment (e.g., control cells with little or no neurite outgrowth). For plate reader quantification it is advisable to also have a cell-free control for subtracting fluorescence background. 

Media and Reagents

Special Tools

• Fluorescence microscope
• Fluorescence microplate reader (bottom-read only)

Note:Standard FITC or fluorescein filter settings work well for the green fluorescent Cell Viability Indicator whereas standard TRITC or Cy3 filter settings are suitable for the orange fluorescent Cell Membrane Stain.

For further guidance, refer to the protocol provided with the Neurite Outgrowth Staining Kit.

Live-cell staining protocol

1. Remove the medium from the cultures to be stained. Optional: rinse the cultures to be stained with D-PBS and aspirate.
2. Apply an appropriate volume of the 1X working Stain Solution to each well or dish to be assayed.
3. Incubate the cultures for 10 – 20 minutes at room temperature or 37 °C.
4. Remove the stain and discard it. Optional: rinse the cultures with D-PBS and aspirate.
5. Apply an appropriate volume of the 1X working Background Suppression Solution to each well or dish to be assayed.
6. Analyze the sample under a fluorescence microscope or using a fluorescence microplate reader.
7. Optional: following analysis, replace the Background Suppression Solution with fresh cell culture medium and continue to maintain the culture. Stained cells may be re-stained as early as 24 hours after the initial staining.

Fix and stain protocol

If cell fixation is desired, the 1X working Stain Solution should have been prepared in buffer containing 4% paraformaldehyde.

1. Remove the medium from the cultures to be stained. Optional: rinse the cultures to be stained with D-PBS and aspirate.
2. Apply an appropriate volume of the 1X working Stain Solution (prepared in buffer containing 4% paraformaldehyde) to each well or dish to be assayed.
3. Incubate the cultures for 10 – 20 minutes at room temperature or 37 °C.
4. Remove the stain and discard it. Optional: Rinse the cultures with D-PBS and aspirate.
5. Apply an appropriate volume of the 1X working Background Suppression Solution to each well or dish to be assayed.
6. Analyze the sample under a fluorescence microscope or using a fluorescence microplate reader.

Figure 1 - Visualizing and quantifying cell viability and neurite outgrowth using the dual-color Neurite Outgrowth Staining Kit. (A) Cryopreserved primary rat cortex neurons (Cat. no. A1084002) were thawed and plated in 96-well format and grown in Neurobasal medium (Cat. no. 21103049) supplemented with B-27 Serum-free Supplement (Cat. no. 17504044) or B-27 Plus Supplement (Cat. no. A3582801) for 7 days prior to staining with the Neurite Outgrowth Staining Kit. Left, image from using the kit’s green fluorescent Cell Viability iIndicator, which primarily stains the cell bodies of living cells. Right, image from using the kit’s orange fluorescent Cell Membrane Stain in the same field of view, which stains the membranes of neurite extensions in addition to cell bodies. (B) PC12-derived Neuroscreen™-1 cells were treated with a serial dilution of nerve growth factor (NGF) for 4 days prior to measuring relative cell viability and neurite outgrowth using a plate reader (left panel) and taking images of representative wells (right panel). The Neurite Outgrowth Staining Kit simultaneously measures cell viability (green fluorescence), which was unchanged in this experiment, and relative neurite outgrowth (orange fluorescence) which increased in an NGF dose-dependent manner. 

 

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Fluorescence Imaging Filter Settings

Fluorescence Plate Reader Filter Settings - Monochromator-based

Important! Only bottom-read mode should be used with cells stained in clear-bottom microplate.

Fluorescence Plate Reader Filter Settings - Filter-based

Important! Only bottom-read mode should be used with cells stained in clear-bottom microplate.