Jul 10, 2023
Fura-2 imaging of ionomycin response, with and without R568; a CaSR positive modulator
- 1University of Oxford
Protocol Citation: Peter Kilfeather 2023. Fura-2 imaging of ionomycin response, with and without R568; a CaSR positive modulator. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg39p4zg25/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 18, 2022
Last Modified: July 10, 2023
Protocol Integer ID: 71468
Funders Acknowledgement:
ASAP
Medical Research Council
Abstract
Fura-2 imaging protocol to accompany Kilfeather, Khoo et al., 2023: Single cell spatial transcriptomic and translatomic profiling of dopaminergic neurons in health, ageing and disease
Data Capture
Data Capture
Prepare Fura-2 AM to 5 mM concentration in calcium-free HBSS supplemented with 20 mM HEPES.
Dilute Fura-2 AM to the final concentration of 2.5 mM in Neurobasal medium supplemented with B27 and L-glutamine, with DMSO (0.1%) or the CaSR positive modulator R568 (10 mM).
Incubate iPSC-derived dopaminergic neurons (DAns) in the solution of Fura-2 AM and drug for 1 h at 37°C, 5% CO2 and then image on a FlexStation 3 Multi-Mode Microplate Reader (Molecular Devices) at 37°C.
Excite the dye at 340 nm and 380 nm and detect at 510 nm. Image each well every 4 s for 100 s and inject with ionomycin (final concentration 5 mM) after a baseline of 28 s.
Analysis
Analysis
For the analysis, compute the 340/380 ratio, and then subtract the baseline from all the timepoints, to obtain a normalised trace for each well. Find the maximum intensity (peak amplitude) of the normalised trace and calculate the area under the curve (AUC) using the left rectangular approximation method.