Amicon ultra 4 10 kda centrifugal filter units

To analyse the effect of myelin on TREM2, primary microglia were treated with myelin (5 or 30 μg/mL) or LPS (1 μg/mL) for 24 hours. Cells were collected and resuspended in RIPA buffer (150 mM NaCl, 1.0% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0). Cell lysates were cleared of cellular debris by centrifugation (15,000 r.p.m. for 5 minutes), and equal amounts of protein were separated by SDS-PAGE on 12.5% acrylamide gels and transferred to 0.45-μm nitrocellulose membranes. The membranes were blocked (3% milk and 0.05% Tween 20 in TBS), incubated with primary antibodies overnight at 4 degrees in blocking solution and washed with TBST 3 times. The membranes were then incubated with horseradish-peroxidase-conjugated secondary antibodies for 1 hour at room temperature. After being washed in TBST, the membranes were visualized using an enhanced chemiluminescence system (32106, Pierce). The antibodies used were anti-TREM2 (1:50, clone 5F4) and anti-tubulin (1:10,000, Cat. No. T6557, Sigma-Aldrich). To measure the amount of soluble TREM2, the medium of the treated cells was collected and centrifuged at 3,200g for 20 minutes to remove cell and myelin debris. The medium was then concentrated with Amicon Ultra-4 10 kDa Centrifugal Filter Units (UFC801024, Merck), and equal volumes were loaded on a 12.5% acrylamide gels and run as described above.