This article presents a carefully described technique with some tips on successful imaging of calcium with Fura-2 AM in the ventral spinal motor neurons of the spinal cord.
1. Preparing Enriched Cultures Containing Motor Neurons, from the Ventral Spinal Cord of 13-Day Old Mouse Embryos, to Obtain Culture Adherent Cells
The first step is the centrifugation of motor nerve cells and glial cells, on a 6.2% OptiPrep cushion.
Then the glial cells were plated on poly-L-ornithin (1 mg/mL in borate buffer) to prepare the glial feeder layers, using coated 12 mm dishes from Marienfeld GmbH & Co. KG, Germany. The density used was 50,000 cells/dish and the medium employed was DMEM/Ham's F12-medium, to which fetal calf serum (10%) was supplemented over the course of the first week, to be replaced later with horse serum (10%) and penicillin (10 U/mL)/streptomycin (10 μg/mL).
The enriched fraction containing motor neurons was then implanted as seeds on the already prepared glial feeder layers, at a density of 30,000 cells/dish. Now the calcium imaging experiments were carried out.
The temperature at which the cultures were maintained was 37 °C, within a 5% CO-humidified incubator. The experiments were begun at day 13 of the culture’s in vitro life.
2. Dissolve Fura2-AM
In this step the Fura-2 AM is directly dissolved in the vial using DMSO to obtain a final concentration of 2 mM. Keep the solution protected from light by covering it with aluminum foil. Prepare 2 µl aliquots and keep them in the dark. Be aware that smaller volumes will bleach faster.
3. Add Fura-2 AM to Your Cells
Now 2 µl Fura-2 AM aliquots are dissolved in each 500 µl well for a final concentration of 8 µM. This was achieved by adding Fura-2 AM straight to the culture medium containing motor neurons, which is made using neurobasal medium, B27 neuromix (2%), nitrogen supplement (0.2%), L-glutamine (1 mM), horse serum (2%), penicillin (10 U/mL)/ streptomycin (10 μg/mL) and BDNF (2 ng/mL).
The aliquot should be prewarmed (using just the heat from your hands) to enable proper dissolution to take place. Otherwise, small dye aggregates will be seen as bright specks during imaging.
Put the cells back into incubator, incubate for 20 minutes.
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.). It is important to make sure that all solutions are prewarmed.
6. Image Cells
Now the coverslip should be put in the imaging chamber through which extracellular solution flows constantly.
The cells are imaged using alternate 350 nm and 380 nm excitation at 5 ms exposure, and the light is collected through an LP 440 filter.
Now the glial cells show spontaneous regular calcium waves.
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.
This protocol was kindly submitted by Dr Janin Lautenschläger (Lautenschläger et al. Exper Neurol 2013, PMID: 23578819).
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