Measuring Neuronal Cell Health through Viability and Neurite Outgrowth

Introduction

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

  • Neurite Outgrowth Staining Kit (Cat. no. A15001)
    • Cell Membrane Stain
    • Cell Viability Indicator
    • Background Suppression Dye
  • Dulbecco’s Phosphate-Buffered Saline (D-PBS) (Cat. no. 14287) or Hank's Balanced Salt Solution (HBSS) (Cat. no. 14025)
  • Optional: 4% paraformaldehyde in buffer for fixing cells (e.g., Cat. no. R37602

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.

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Preparing Reagents

Prepare a fresh 1X working Stain Solution and a separate 1X Background Suppression Solution using the reagents provided in the Neurite Outgrowth Staining Kit as follows:
  1. Thaw the reagents and ensure that they are completely in solution before proceeding. Note that the Background Suppression Dye can also be stored at room temperature. 
  2. Prepare a fresh 1X working Stain Solution by diluting the Cell Viability Indicator (1000X) and the Cell Membrane Stain (1000X) in sterile, tissue culture-grade D-PBS (or buffer containing 4% paraformaldehyde if cell fixation is desired). Mix well. Prepare enough 1X stain to completely cover each well or dish to be assayed.
  3. Separately, prepare a fresh 1x working Background Suppression Solution by diluting the Background Suppression Dye (100X) in sterile, tissue culture-grade D-PBS. Mix well. Prepare enough 1X solution to completely cover each well or dish to be assayed.

    The resulting Stain Solution and Background Suppression Solution are ready to be used. The final concentration of DMSO in the Stain Solution is ≤ 0.2%, a level generally innocuous to most cells.
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Methods

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) 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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Instrument Filter Settings

Fluorescence Imaging Filter Settings

Dye Excitation (nm) Emission (nm)
Cell Viability Indicator495515
Cell Membrane Stain555565

Fluorescence Plate Reader Filter Settings - Monochromator-based

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

Dye Excitation (nm) Emission (nm) Bandwidth (nm)
Cell Viability Indicator48352512
Cell Membrane Stain5545675

 

Fluorescence Plate Reader Filter Settings - Filter-based

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

Dye Excitation (nm) Emission (nm) Bandwidth (nm) Dichroic mirror (nm)
Cell Viability Indicator480520 or 53525 or 30510 or 50:50 band splitter
Cell Membrane Stain531 or 535579 or 59025 or 20555 or 560

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For research use only. Not for use in diagnostic procedures.

B-27® is a registered trademark of Southern Illinois University.