Vala Assay: #NT2
Cell Types: Human glutamatergic neurons derived from hiPSCs in co-culture with human microglia derived from hiPSCs
Main Goal of Assay: Determine the effects of test compounds on calcium transient activity in co-cultures of human glutamatergic neurons and human microglia, both derived from induced pluripotent stem cells. Neurons exhibit action potential-dependent and -independent peaks in intracellular calcium concentration that are dysregulated in aging, traumatic brain injury, and neurodegenerative diseases. Microglia, the main central nervous system immune cells, also exhibit transient increases in intracellular calcium concentration, especially under pathological conditions.
This assay measures the fluorescence of calcium indicator Rhod-4 at 4 Hz for 2 minutes to capture neuronal and microglial calcium transient activity on a cell-by-cell basis. After live imaging, the cells are fixed, immunolabeled, and imaged for cell-specific markers (e.g., βIII tubulin for neurons and IBA1 for microglia). Vala’s CyteSeer® image analysis program first aligns the live and immunofluorescence images to confirm the identity of each cell. CyteSeer® then reports a range of parameters (percent of active cells, event frequency, mean and maximum peak amplitudes, peak width, etc.) to provide a comprehensive picture of how each compound affects neuronal and microglial calcium activity.
CyteSeer® Data Readout
- Fluorescence channel #1 (DAPI): Hoechst (cell number, viability)
- Fluorescence channel #2 (green channel, βIII-tubulin): neuron number and morphology
- Fluorescence channel #3 (red channel): Rhod-4 (calcium transient parameters)
- Fluorescence channel #4 (far red channel, IBA1): microglia number and morphology