A detailed protocol with tips and tricks on how to image calcium using Fura-2 AM in motor neurons from ventral spinal cord.
1. Culture adherent cells - motor neuron-enriched cultures from ventral spinal cord (13-day old mouse embryos)
Motor neurons and glial cells were purified by centrifugation on a 6.2% OptiPrep cushion.
Glial feeder layers were prepared by plating the glial cells on poly-L-ornithin (1 mg/mL in borate buffer) coated 12-mm dishes (Marienfeld GmbH & Co. KG, Germany) at a density of 50,000 cells/dish in DMEM/Ham's F12-medium supplemented with foetal calf serum (10%) for the first week, later on with horse serum (10%) and penicillin (10 U/mL)/streptomycin (10 μg/mL).
Motor neuron enriched fraction was seeded on these pre-established glial feeder layers at a density of 30,000 cells/dish for calcium imaging experiments.
Cultures were kept in a 5% CO-humidified incubator at 37 °C and used for experiments starting on day 13 in vitro.
2. Dissolve Fura2-AM
Dissolve Fura-2 AM directly in the vial using DMSO (final concentration 2 mM).
Protect from light with aluminum foil.
Aliquot to 2 µl and store in the dark. Caution: Beware that small volumes will bleach faster.
3. Add Fura-2 AM to your cells
Dissolve 2 µl Fura-2 AM aliquot per 500 µl well (final concentration of 8 µM) = Fura-2 AM was added directly to the motor neuron culture medium, consisting of neurobasal medium, B27 neuromix (2%), N2 supplement (0.2%), L-glutamine (1 mM), horse serum (2%), penicillin (10 U/mL)/ streptomycin (10 μg/mL) and BDNF (2 ng/mL).
Tip: Prewarm the aliquot (using just the heat from your hands), otherwise it does not dissolve properly and you can see small bright dye aggregates during imaging.
Put cells back into incubator, incubate for 20 min.
5. Take out coverslip and transfer to imaging setup
Put coverslip in a small Petri dish containing standard extracellular solution containing (in mM) HEPES 11.6, NaCl 129.1, KCl 5.9, glucose 11.5, MgCl 1.2, CaCl 3.2 and adjusted to pH 7.4 with NaOH. (This already washes the coverslip.)
Make sure that all solutions are prewarmed.
6. Image cells
Put coverslip in your imaging chamber, continuously perfused by extracellular solution (see above).
Image cells by alternate excitation at 350 nm and 380 nm (5 ms exposure), collect light through a LP 440 filter.
Spontaneous calcium waves can be seen regularly in glia cells.
Motor neurons also show spontaneous calcium transients, but only when neuronal networks are very dense.
Motor neurons show calcium transients upon stimulation with kainate, a AMPA receptor agonist.