HCS image with synaptogenesis features shown as an overlay

Optimized, automated assays for HCS

This automated assay uses the co-localization of presynaptic and postsynaptic markers to identify synapses and correlate them with neuronal and neurite morphology. Neuronal morphology is captured using a nuclear stain to locate the cell and a choice of stains or immunofluorescent labels to detect the cell body and neurites. Synapses are located using two channels for co-localization and immunofluorescent detection with antibodies for presynaptic (e.g., synaptophysin) and postsynaptic (e.g., PSD95) markers.

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Typical synaptogenesis assay

Fixed-cell immunofluorescent assay protocol.

Imaging mode

  • Widefield or confocal
  • 20x magnification

Automatically measured properties

  • Neurite count
  • Neurite length
  • Neurite branching
  • Presynaptic vesicles
  • Postsynaptic spots
  • Synapse number

Since analysis is performed during image acquisition, it is possible to acquire an optimized data set to ensure the number of selected neurons to satisfy your statistical needs.

Synaptogenesis assay images

Three-panel HCS synaptogenesis assay image showing stained cells and software overlays

Syantogenesis assay. (Left) A hippocampal primary neuron preparation was labeled using Invitrogen Hoechst 33342 stain (blue) and an Invitrogen Alexa Fluor 488 MAP2 antibody conjugate. (Center and Right) Synaptogenesis features were automatically identified using Thermo Scientific HCS Studio Software. Cell bodies are identified in blue, and spots are identified in pink. Neurites are identified in green, and overlaps between spots and neurites are labeled in yellow.


Sample experimental data

Four-panel graph of different synaptogenesis assay parameters

Dose-response plots showing the effect of glutamate and kainite concentration on rat hippocampal neurons using automated measurements of presynaptic intensity, postsynaptic intensity, vesicle counts, and synapse counts.

Resources

Instrument systems

HCS reagents optimized for use with your HCS platform

HCS application notes