Fluo-4 Calcium Imaging
Edinson Lucumi Moreno
Abstract
This protocol details the measurement of the firing activity of cultured iPSC derived neurons. This procedure is used to prepare cultured neurons for Calcium imaging.
Attachments
Steps
Procedure for Calcium Fluo-4
Prepare 1millimolar (mM) Fluo-4 stock solution by adding 45µL of DMSO to vial.
Prepare 5µL aliquots of 1millimolar (mM) Fluo-4 stock in PCR Eppendorf tubes and store them at -20°C.
Take one 5µL aliquot of 1millimolar (mM) Fluo-4 and transfer to 995µL of neurobasal medium (work at low light).
Transfer medium from selected wells of the 96 well plate with differentiated neurons to an Eppendorf tube.
Wash well by adding 150µL of neurobasal medium, be careful not to detach of perturb the neurons.
Repeat the washing step 1 more time.
Add 120µL of Fluo-4 AM [5micromolar (µM) in neurobasal medium] into wells.
Incubate for 0h 20m 0s.
Remove the neurobasal medium with Fluo-4 AM, by transferring it to a waste container.
Wash well by adding 150µL of neurobasal medium, be careful not to detach of perturb the neuron.
Remove the neurobasal medium to waste.
Add the old differentiation medium to the same well you took them from.
Incubate for 0h 10m 0s.
Acquire time lapse images in Nikon microscope using the Fluo-4 Calcium imaging acquisition protocol.
