Comprehensive Method for Culturing Embryonic Dorsal Root Ganglion Neurons for Seahorse Extracellular Flux XF24 Analysis

Abstract

Changes in mitochondrial dynamics and function contribute to progression of multiple neurodegenerative diseases including peripheral neuropathies. The Seahorse Extracellular Flux XF24 analyzer provides a comprehensive assessment of the relative state of glycolytic and aerobic metabolism in live cells making this method instrumental in assessing mitochondrial function. One of the most important steps in the analysis of mitochondrial respiration using the Seahorse XF24 analyzer is plating a uniform monolayer of firmly attached cells. However, culturing of primary dorsal root ganglion (DRG) neurons is associated with multiple challenges, including their propensity to form clumps and detach from the culture plate. This could significantly interfere with proper analysis and interpretation of data. We have tested multiple cell culture parameters including coating substrates, culture medium, XF24 microplate plastics, and plating techniques in order to optimize plating conditions. Here we describe a highly reproducible method to obtain neuron-enriched monolayers of securely attached dissociated primary embryonic (E15) rat DRG neurons suitable for analysis with the Seahorse XF24 platform.

Keywords: Seahorse XF24 Extracellular Flux analysis; embryonic dorsal root ganglion neurons; extracellular acidification rate; mitochondrial respiration; oxygen consumption rate.

Figure 1

Elimination of non-neuronal cells in DRG neuronal cultures. Although Thy 1.1 pre-plating alone did not completely remove non-neuronal cells (1 DIC, yellow arrows; red arrows, DRG neurons), it significantly reduced the amount of time DRG neurons spent in anti-mitotic media. Dissociated DRG neurons seeded at 50,000 cells/well in a 24-well Seahorse XF24 polystyrene microplate at 1 and 7 DIC at 5× phase objective. Scale bar: 200 μm.

Figure 2

Optimization of coating substrates for DRG neuronal culture. (A) 24-well Seahorse XF24 microplates coated with collagen promoted aggregation and clumping of DRG neurons (yellow asterisks). (B) Poly-lysine, poly-ornithine, and the optimal substrate combination of poly-lysine + 10% collagen provided equally satisfactory DRG neuron monolayers. (C) Detachment of DRG neurons cultured on poly-lysine and poly-ornithine alone or in combination with laminin observed after 6 DIC. DRG neurons seeded at 50,000 cells/well in a XF24 polystyrene microplate imaged at 7 DIC with a 5× phase objective. Scale bar: 200 μm.

Figure 3

Seahorse XF24 mitochondrial stress analysis. Using the optimal seeding density (50,000 cells/well) and coating substrate combination (poly-lysine + 10% collagen) on 24-well Seahorse XF24 polystyrene microplate, well-adhered, and dispersed DRG neuronal cultures were maintained throughout the culture process and Seahorse XF24 analysis (7 DIC). Each data point is presented as mean ± SEM. Injection series: (A) Oligomycin (1.5 μM); (B) FCCP (0.75 μM); (C) rotenone (1.0 μM) and antimycin A (1.0 μM).

Figure 4

Seahorse XF24 optimization of DRG neuron seeding density. Titration of DRG neuron seeding density with the XF24 platform maximizes increases and decreases in OCR and ECAR. Linear increases are seen in both (A) OCR and (B) ECAR spectra with increasing cell number. (A–D) XF24 microplates seeded at 25,000 cells/well (purple line) produced negligible OCR and ECAR due to detachment either before or during analysis. (D) ECAR values plateau between 40,000 (red line) and 50,000 (green line) cells/well indicating the optimal seeding density for Seahorse XF24 analysis is likely 40,000 cells/well. After Seahorse XF24 mitochondrial stress analysis, XF24 microplates seeded at 40,000 cells/well showed minor detachment, thus, 50,000 cells/well was selected as the optimal density. Each data point is presented as mean ± SEM. All readings completed on polystyrene XF24 microplates pre-coated with 0.1 mg/ml poly-lysine + 10% collagen. Injection (A) FCCP (0.5 μM).

Figure 5

“Edge-effect.” DRG neurons cultured in 24-well Seahorse XF24 microplates at RT display uneven distribution when placed directly in a humidified incubator at 37°C and 5% CO₂. This “edge-effect” is caused by a thermal gradient between the periphery and center of each well. DRG neuronal culture at 1 DIC seeded at 50,000 cells/well of an XF24 polystyrene microplate at 5× phase objective. Scale bar: 200 μm.