This protocol provides an overview of the IncuCyte Fluorescence Neurite Analysis Assay methodology. It is compatible with the IncuCyte ZOOM® live-cell analysis system using your choice of neuronal cells and treatments. The IncuCyte NeuroLight™ Red labelling reagent is used to fluorescently label living neurons without perturbing cell function or biology. The NeuroLight Lentivirus reagent encodes a red fluorescent protein driven off a synapsin promoter to ensure minimal expression in non-neuronal cell types. The highly flexible assay format can be combined with the IncuCyte™ Annexin V Green reagent or IncuCyte™ Cytotox Green reagent for multiplexed measurements of cytotoxicity and apoptosis.
1) Coat a 96-well plate with the recommended coating for your neuronal cell type of choice (see table below).
|Neuronal cell type||Subtype or supplier||Plate Coating||Coating protocol|
|Immortalized neuronal cell lines||Neuro2A or SH-SY5Y||Poly-D-lysine||DL (100 µg/ml) solution incubated overnight, aspirated, washed with sterile water, and incubated at room temperature for 1h|
|Primary neurons||CNS||Poly-D-lysine||PDL (100 µg/ml) solution incubated overnight, aspirated, washed with sterile water, and incubated at room temperature for 1h|
|PNS||Poly-D-lysine + laminin||PDL coated as described above followed immediately by Laminin (3.3 µg/ml) solution incubated for 2 h and aspirated just prior to seeding|
|iPSC-derived neurons||CDI (iCell or iDopa)||Poly-D-lysine + laminin||PDL coated as described above followed immediately by Laminin (3.3 µg/ml) solution incubated for 2 h and aspirated just prior to seeding|
|Axiogenesis (Dopa4U or Peri 4U)||Poly-L-ornithine + laminin||PLO (0.1%) solution incubated for 1 h, aspirated and washed followed immediately by Laminin (3.3 µg/ml) solution incubated for 2 h and aspirated just prior to seeding|
1) Plate neurons at 15,000 to 30,000 cells/well (100 µL per well) in your chosen culture media.
2) After 4 hours add the IncuCyte NeuroLight Lentivirus reagent at desired multiplicity of infection (MOI = TU/cell) diluted in neuronal culture media (100 µL/well). An MOI of 0.3 to 3 is recommended for most neuronal cell types, however, an optimized MOI should be determined for each cell type used.
3) Incubate at 37°C, 5% CO2 for 24 hours.
4) Remove lentivirus from all wells by aspirating 190µL and adding back 140µL fresh culture media.
5) Plate Astrocytes 15,000 cells/well (50 µL/well) in your chosen astrocyte culture media
6) Return plate to incubator for an additional 24-48 hours, monitoring expression using an IncuCyte® live cell analysis system. Scan plate every 4-6 hours using the phase contrast and red fluorescence channels.
7) OPTIONAL: Add Uridine + 5-FDU at a final assay concentration of 8 µg/mL and 28 µg/mL respectively to inhibit astrocyte proliferation by performing a 50% media exchange (100 µL/well).
8) Replace media every 3 days for the duration of the assay by performing a 50% media exchange with neuronal culture media.