Microcon centrifugation filters

Animals were deeply anaesthetised with sodium pentobarbital (Euthatal^®, 80 mg/kg, administered intraperitoneally) and transcardially perfused with sterile PBS supplemented with EDTA (12.5 mM). An 8 mm section of contused injury epicentre spinal cord (or equivalent intact control) was collected at 6, 12 h and at 1, 3 and 7 days after contusion and snap-frozen in liquid nitrogen. Spinal cords were bisected while frozen, in order to conduct both protein and RNA analysis (described below). For protein analysis, tissue was homogenised with lysis buffer on ice with a tissue homogeniser (Cole-Parmer, LabGEN 7 Series Homogeniser). Samples were centrifuged (20,000 × g, 4 °C, 20 min), the supernatant recovered, and protein quantified using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Samples were concentrated to 4 mg/ml using MicroCon centrifugation filters (Millipore) to ensure equal amounts of protein. Cytokine protein levels were then analysed using the MILLIPLEX MAP Rat 27 Cytokine & Chemokine magnetic bead panel (RECYMAG65K27PMX—Millipore) on a Luminex (Millipore) as per manufacturer’s protocol.

Adult female C57/Bl6 mice were perfused with sterile saline and a 5-mm-long segment of sham or the contused spinal cord was collected at 4 and 72 h after SCI (n = 3 per group and time point) and snap-frozen. The spinal cords were homogenized, and protein concentration was determined using the DC Protein Assay (Bio-Rad). Samples were concentrated to 4 mg/ml using MicroCon centrifugation filters (Millipore) to ensure equal amounts of protein. Cytokine protein levels were then analyzed using the Milliplex MAP Mouse Cytokine/Chemokine magnetic bead panel (Millipore) on a Luminex (Millipore) as per the manufacturers’ protocol.