PC12 Cell Neurite Analysis

Introduction

Facing an aging society, pathological investigation and effective therapy development are urgently required for neurodegenerative diseases which could often relate to aging, such as amyotrophic lateral sclerosis, Alzheimer’s disease, Parkinson’s disease and Huntington’s disease. Especially in the neuroscience research field, studies have been progressed rapidly so as to clarify the involvement of various signal transduction pathways in the progress of neurodegenerative disease. Neurons consist of soma containing nucleus, dendrite for receiving signals and axon for outputting them. Their fundamental functions involve receiving input stimuli, generating action potential and transmitting information to other cells. The very minute space between the axon terminal and the dendrite is filled with the structure to transmit chemical or electrical information, called synapse. In neuroscience studies, such indicators as action potential of sodium and calcium ions and localization and quantity of various signal transmission substances, are used to screen pharmacologically active substances. Our “Neurite Analysis” protocol can provide numerical data for morphological changes in nerve fibers and the localization and behavior of proteins on nerve fibers, which enables analysis of cytotoxicity studies on cultured neurons. The following is an example of the analysis of neurite extension following the stimulation by a nerve growth factor (NGF) in PC12 cells, a model used for the study of neuronal differentiation and neuro-regeneration.

Fig.1 Images acquired using the CellVoyager CV8000
(a) Original image before NGF stimulation

(b) Processed image from (a) (Recognized nuclei and β-Tubulin III signals)

(c) Original image after NGF (111ng/ml) stimulation

(d) Processed image from (c) (Recognized nuclei and β-Tubulin III signals)
All of the above images are superimpose of nuclei (Hoechst33342) and β-Tubulin III(Alexa488) images.
Red lines are the neurites recognized by the “Neurite Analysis” protocol.

Fig.2 NGF concentration and neurite changes
(a) Dose-response curve of the neurite number per cell

(b) Distribution of neurite length in individual cell by NGF dosage

Experiment procedure

1. PC12 cells were seeded on in 96-well plates coated with type IV collagen at 5,000 cells/well, and cultured for 24 hours.
2. The cells were cultured in serum-free medium for another 24 hours.
3. The cells were treated with NGF (final concentration: 0 - 111ng/ml) for three days for reaction.
4. The cells were fixed with formaldehyde, immunostained β-Tubulin III with Alexa488, and stained nuclei with Hoechst33342.
5. Images were acquired using the CellVoyager CV8000 equipment under the following conditions:
   •Magnification: 20x objective lens
   •Images captured per well: 4 Fields
   •Exposure time: Alexa488: 300msec, Hoechst33342: 100msec
6. The acquired images were analyzed using the “Neurite Analysis” protocol.
   •Nuclei regions were recognized from the nuclei images.
   •Soma regions and neurite regions were recognized from the β-Tubulin III images.
   •The length, quantity and branching number of neurite were calculated.

Results and Conclusion

Neurite outgrowth due to NGF treatment were analyzed, using the CellVoyager CV8000 images and “Neurite Analysis” protocol. As a result, numerical data for the increase in neurite number depending on NGF concentration were obtained (Fig. 2a). Although not shown here, similar dose-response curves were obtained for the length and branching number of neurite. Yokogawa analysis software also allows for easy output of numerical data for the length, quantity and branching number of neurite in individual cells, thus enables creation of graphs for individual cell data (Fig. 2b). In this way, the “Neurite Analysis” protocol is an efficient tool for easy acquisition of various neurite information by a cell or by a well, which lead to detailed evaluation of the effect of compounds on neurodegenerative diseases.

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