IncuCyte® Fluorescence Neurite Analysis Assay General Protocol

IncuCyte® Fluorescence Neurite Analysis Assay General Protocol

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.

Neuronal co-cultures—fluorescence protocol overview

neurite-protocol-figure_2


General Protocol

Day -1

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

Day 0

1) Plate neurons at 15,000 to 30,000 cells/well (100 µL per well) in your chosen culture media.

Day 0 + 4 hours

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.

Day 1

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.

Day 3

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).

Days 6, 9, 12

8) Replace media every 3 days for the duration of the assay by performing a 50% media exchange with neuronal culture media.

  • OPTIONAL: Prepare treatments at 2X final assay concentration and add at the time of media exchange (100 µL/well).
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