Imaging Calcium in Motor Neurons Using Fura-2 AM

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.

4. Incubate

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.

Reference

This protocol was kindly submitted by Dr Janin Lautenschläger (Lautenschläger et al. Exper Neurol 2013, PMID: 23578819).

About Abcam

Abcam is a global life sciences company providing highly validated antibodies and other binders and assays to the research and clinical communities to help advance the understanding of biology and causes of disease.

Abcam’s mission is to serve life scientists to help them achieve their mission faster by listening to their needs, continuously innovating and improving and by giving them the tools, data and experience they want. Abcam’s ambition is to become the most influential life science company for researchers worldwide.


Sponsored Content Policy: News-Medical.net publishes articles and related content that may be derived from sources where we have existing commercial relationships, provided such content adds value to the core editorial ethos of News-Medical.Net which is to educate and inform site visitors interested in medical research, science, medical devices and treatments.

Last updated: May 22, 2019 at 7:58 AM

Citations

Please use one of the following formats to cite this article in your essay, paper or report:

  • APA

    Abcam. (2019, May 22). Imaging Calcium in Motor Neurons Using Fura-2 AM. News-Medical. Retrieved on August 22, 2019 from https://www.news-medical.net/whitepaper/20190522/Imaging-Calcium-in-Motor-Neurons-Using-Fura-2-AM.aspx.

  • MLA

    Abcam. "Imaging Calcium in Motor Neurons Using Fura-2 AM". News-Medical. 22 August 2019. <https://www.news-medical.net/whitepaper/20190522/Imaging-Calcium-in-Motor-Neurons-Using-Fura-2-AM.aspx>.

  • Chicago

    Abcam. "Imaging Calcium in Motor Neurons Using Fura-2 AM". News-Medical. https://www.news-medical.net/whitepaper/20190522/Imaging-Calcium-in-Motor-Neurons-Using-Fura-2-AM.aspx. (accessed August 22, 2019).

  • Harvard

    Abcam. 2019. Imaging Calcium in Motor Neurons Using Fura-2 AM. News-Medical, viewed 22 August 2019, https://www.news-medical.net/whitepaper/20190522/Imaging-Calcium-in-Motor-Neurons-Using-Fura-2-AM.aspx.

Other White Papers by this Supplier