Ecl western blotting detection reagents and analysis system

MPCs were seeded in 6-well plates and were differentiated to macrophages. The latter were then subjected to stimulation with the reagents of interest and whole cell lysates were prepared. For this purpose, the cells were scraped into ice-cold PBS with a rubber policeman, pelleted by centrifugation and after resuspension in 4 × Laemmli sample buffer (8% (w/v) SDS, 0.1 M dithiothreitol, 40% (v/v) glycerol, 0.2 M Tris, pH 8.0) supplemented with phosphatase inhibitor cocktail II (Sigma) lysed by sonification (ten pulses) and heating for 5 min at 96 °C. Remaining insoluble debris was removed by centrifugation and the resulting whole cell lysates were subjected to SDS–PAGE. Separated proteins were transferred from the gel to nitrocellulose and after blocking of nonspecific binding sites by incubation in Tris-buffered saline with 0.1% (v/v) Tween 20 and 5% (w/v) dry milk, immunoblotting was performed with the primary antibodies of interest, horseradish peroxidase-conjugated secondary antibodies (Dako, Hamburg, Germany) and the ECL western blotting detection reagents and analysis system (Thermo Fisher Scientific).

Macrophages were stimulated with the reagents of interest and were subsequently scraped into ice-cold PBS with a rubber policeman. Cells were spin down by centrifugation (2 min, Eppifuge full-speed) and resuspended in 4 × Laemmli sample buffer (8% (w/v) SDS, 0.1 M dithiothreitol, 40% (v/v) glycerol, 0.2M Tris, pH 8.0) supplemented with phosphatase inhibitor cocktail II (Sigma, Steinheim, Germany). Cell lysis was enhanced by sonification (ten pulses) and heating (5 min, 96 °C). Lysates were cleared from remaining insoluble debris by centrifugation (8 min, Eppifuge full-speed) and subjected to SDS-polyacrylamide gel electrophoresis. After separation proteins were transferred from the gel to nitrocellulose by wet blotting for 90 min. Remaining free binding sites on the nitrocellulose were blocked by overnight incubation at 4 °C in Tris-buffered saline with 0.1% (v/v) Tween 20 and 5% (w/v) dry milk. After washing once with TBST, the nitrocellulose membrane was incubated with the primary antibodies of interest (4 °C, overnight). After washing twice again in TBST, the nitrocellulose membrane was incubated with an appropriate horseradish peroxidase-conjugated secondary antibody (Dako, Hamburg, Germany) and the antigen–antibody complexes were finally detected by help of the ECL western blotting detection reagents and analysis system (Thermo Fisher Scientific).