This protocol provides an overview of the Incucyte Fluorescence Neurite Analysis Assay methodology. It is compatible with the Incucyte ® Live-Cell Analysis System using your choice of neuronal cells and treatments. The Incucyte® Neurolight Lentivirus is used to fluorescently label living neurons without perturbing cell function or biology. The Neurolight Lentivirus encodes a 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 Dye or Incucyte® Cytotox Dye 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) Add appropriately diluted Neurolight Lentivirus to achieve desired concentration. The final well volume should be 200 μL per well.
Note: Quality control for the Incucyte Neurolight Lentivirus is the ability to efficiently infect Incucyte rCortical Neurons to express the fluorescent protein, driven off of the synapsin promotor of the Incucyte Neurolight Lentivirus, such that a volume of > 3.2 μL/20,000 neurons results in a neurite length of > 50 mm/mm2 in a neurite outgrowth assay (rCortical Neurons/rAstrocytes co-culture experiment). We recommend performing a volumetric titration from 100-0.14 μL for each neuronal cell line evaluated. The lowest concentration that results in the highest neurite outgrowth measurement should be selected. Evaluation of neurite dynamics is to be performed on an Incucyte Live-Cell Analysis System.
3) Incubate at 37°C, 5% CO2 for 24 hours.
4) Remove the Neurolight Lentivirus from all wells by aspirating 190µL and adding back 140µL fresh culture media.
5) Plate astrocytes at a density of 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 fluorescence channel of choice.
7) OPTIONAL: Add Uridine + 5-Fluoro-2′-deoxyuridine 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.