Mouse anti cspg antibody

Thirty hindbrains of HH18 chick embryos were harvested and dissociated into single cells using collagenase type 4 (200 units/ml, Worthington 47B9407). Cells were then centrifuged at 600 g for 10 min, washed in PBS, then centrifuged again. Next, cells were incubated with mouse anti-CSPG antibody (c8053, Sigma-Aldrich) diluted 1:50 in MACS BSA Stock Solution and autoMACS Rinsing Solution (1:20, Miltenyi Biotec) for 75 min at room temperature. Next, cells were centrifuged and washed in PBS twice, then incubated with anti-mouse Alexa-Fluor 488 antibody (1:200, Thermo Fisher Scientific) in autoMACS Running Buffer (Miltenyi Biotec) for 30 min at room temperature. Cells were then washed, centrifuged, resuspended and kept in hESC media overnight at 4°C. Next, cells were washed with autoMACS Running Buffer and stained with DAPI (1:200 in autoMACS Running Buffer) for 5 min at room temperature, then washed again. 1×10⁷ cells/ml were passed to FACS tubes and sorted using an ARIA III FACS (BD Biosciences) into 1 ml autoMACS Running Buffer. The gating was set according to size and granularity using FSC and SSC to capture singlets and remove debris. The cut-off for sorting the positive (CSPG⁺) and negative (CSPG⁻) cells was based on Alexa-Fluor 488 stained or unstained cells, appropriately, with the exclusion of dead and/or damaged DAPI⁺ cells, chosen by manual gating.

Sixteen μg of lumbar spinal cord protein extracts from symptomatic SOD1^G93A male mice were exposed or not to 1 μg of human recombinant ADAMTS-1 (2197-AD-020), ADAMTS-4 (4307-AD-020) or ADAMTS-5 (2198-AD-020; all recombinant proteins were from R&D SYSTEMS, Minneapolis, MN, USA) in the aggrecanase buffer (50 mM TrisHCl, 125 mM NaCl, 5 mM CaCl₂, pH 7.5) within a final volume of 55 μl, for 24 h at 37 °C (N = 4 per condition). The reaction was stopped by heating the samples at 75 °C for 10 min. Twelve μl of each condition of the above preparations were resolved in a 6 % polyacrylamide gel, the membrane probed with the mouse anti-CSPG antibody (1/1000; Sigma-Aldrich), then with the peroxidase-conjugated anti-mouse secondary antibody (1/5000; GE Healthcare life sciences) and finally revealed by ECL detection, as previously described in the Western Blot section.