Fastprep bead beater system

Spinal cords were homogenized in ice-cold lysis buffer (25 mM Tris HCl pH 7.4, 15 mM NaCl, 10 mM NaF, 10 mM Na Pyrophosphate, 2 mM EDTA, 0.2 mM Na₄OV₃, 1 mM PMSF, Protease Inhibitor Cocktail (1 : 200; Sigma Aldrich, UK)) using the FastPrep® bead beater system (MP Biomedicals, USA). The protein concentration of each sample was determined using a BCA assay (Pierce Biotechnology, UK) and 350 μg of lysate was loaded per membrane (Rat Cytokine Array Panel A, R&D Systems, UK), as per manufacturer's instructions. Cytokine levels were visualised and quantified using IRDye® 800CW Streptavidin secondary antibody (1/2000 in assay buffer 6; Li-Cor, UK) and captured on an Odyssey® Infrared Imaging System. Results were analysed using Odyssey 2.1 Software (Li-Cor, UK) and all absorbance values were corrected for background.

The 4 cm² anode pieces were used for total DNA extraction using a DNA Soil Nucleospin kit (Macherey-Nagel, Düren, Germany). The sample lysis step was performed with a FastPrep bead beater system (MP Biomedicals, Illkirch-Graffenstaden, France) at a speed of 6 m/s for 20 s to detach bacteria from the anode. DNA was stored at −20 °C prior to qPCR. qPCR assays for Geobacter and all bacteria count were conducted on a Rotor-Gene 6000 (Corbett Life Science, Sydney, Australia). Each 20 μL reaction contained the following: 10 μL of SensiFAST SYBR No-ROX mix (Bioline), 0.8 μL of each primer (10 μM; Invitrogen, Waltham, MA, USA) (Table S2), 6.4 μL H₂O, and 2 μL template DNA. PCR reactions were subjected to the following cycling parameters: 95 °C for 2 min, then 30 cycles of 95 °C 15 s, 20 s at the annealing temperature, and 72 °C for 25 s. Each assay included triplicate reactions per DNA sample with three standards (containing 7 different concentrations ranging from 1 × 103 to 1 × 109 copies/µL). Quantitation was calculated using Rotor-Gene 6000 Series Software.