IncuCyte® Label Free Neurite Analysis Assay General Protocol

This protocol provides an overview of the IncuCyte Label Free Neurite Analysis Assay methodology. It is compatible with the IncuCyte® Live-Cell Analysis System using your choice of neuronal cells and treatments. The highly flexible assay format can be combined with our IncuCyte® Annexin V Reagents or IncuCyte® Cytotox Reagents for multiplexed measurements of apoptosis and cytotoxicity.

Neuronal mono-cultures—label-free protocol overview

Protocol

Day 0

1) Coat a 96-well plate with the recommended coating for your neuronal cell type of choice (see table below). NOTE: We recommend use of 96-well plates from TPP (TP92096) as they have high optical clarity and few scratches enabling clear visualization and analysis of fine neurite structures under phase contrast illumination.

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
Primary neurons	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
iPSC-derived neurons	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

Day 1

1) Plate neurons:

Aspirate the coating solution and wash the plate twice with 150 µL of sterile water. Allow to dry for one hour with the lid removed in a culture hood.
Plate neurons at 15,000 to 30,000 cells/well (100 µL per well) in your chosen culture media.
OPTIONAL: If studying treatments that modulate neurite outgrowth add test agents at 3X the final assay concentration (50 µL per well).

2) Image the plate in the IncuCyte® Live-Cell Analysis system with a 20x objective (9 images per well) or 10x objective (4 images per well) using the NeuroTrack Scan Type. Image each well with the phase contrast channel every 6 to 12 hours until the assay is complete (3 to > 10 days).

Days 4, 7, 10

1) Replace media every 3 days for the duration of the assay by performing a 50% media exchange.

OPTIONAL: If studying treatments that modulate neurite network disruption add test agents to previously untreated wells at 3X the final assay concentration (50 µL per well).