Neurite Outgrowth & Mitosis Assay Cell Line
A novel green fluorescent SH-SY5Y cell line has been developed through stable transfection with tubulin tagged Evrogen TagGFP2. This cell line expresses green fluorescent tubulin marking the cell cytoskeleton and it can be easily differentiated using standardized protocols to promote the neurite outgrowth.Cells were then grown in the presence of G418. Single cells with strong green fluorescence were selected by flow cytometry, and allowed to expand. These cells constitutively express the TagGFP2-tubulin fusion protein.
Assay Details: The growth of neurites in human neurons is a critical event in neuronal development, formation and remodeling of synapses, response to injury, and regeneration. Many neuroscience projects are focused on identifying new drugs that affect this event. The discovery of new compounds that can positively affect neuritogenesis would be very important for developing new therapeutics against both neurodegenerative diseases and injury. Measurement of neurite outgrowth using an automated image-based assay can be of use in the research, screening and validation phases of the drug discovery process. Moreover, a neurite outgrowth assay can also be used for testing toxic neuropathies. Recombinant SH-SY5Y cells stably expressing human tubulin TagGFP2 are intended to be use as in vitro model for neuronal differentiation studies, designed to assay for compounds that positively affect neuritogenesis quantifying the number and length of neurites.
Fig 1: SH-SY5Y stably expressing human tubulin tagged in the C-terminus with TagGFP were stimulated with 10 μM of retinoic acid during 7 days. After 3 and 7 days, cells were analyzed with the Becton Dickinson Pathway 855 High Content Bioimager using the Neurite Outgowth application of the Attovision Software. When cells were treated with the retinoic acid, we observed an increase in the length of the developed neurites. The activity was calculated as an increment of neurite length.
Fig 2: Mitosis Assay:(Top) TagGFP2-tubulin without (A) and with 0.1 μM (B) Paclitaxel. (Bottom) Mitotic spindle quantification after 1 hour oftreatment. In this curve, the Ec50 for Paclitaxel was 22.5nM. This assay was validated with an average of Z’= 0.60+/- 0.01 for High Content Screening.