Coomassie protein assay

After CRH stimulation, purified splenic B cells were suspended in NP-40 cell lysis buffer (150 mM sodium chloride, 50 mM Tris pH 8.0, 1% NP-40) and protein extraction was performed in the presence of a complete protease and phosphatase inhibitor mixture (Roche Molecular Biochemicals, Mannheim, Germany). Protein concentrations were determined using a Coomassie protein assay (Bio-Rad, Ivry sur Seine, France).

Caspase 3 activity was analyzed as described previously [25 (link)]. Isolated cells were washed twice with PBS (5 min, 250 g, 4°C) and lysed (50 mM HEPES; 5 mM CHAPS; 5 mM DTT) on ice for 20 min and centrifuged (15 min, 15 000 g, 4°C). Lavage fluid was diluted 1 : 1 with 2x concentrated lysing buffer. The proteins present in supernatants were quantified using Coomassie® Protein Assay (Bio-Rad, USA) and diluted to an equal concentration. 5 μg of protein samples was incubated in an assay buffer in parallels (20 mM HEPES; 2.5 mM CHAPS, 5 mM DTT, 2 mM EDTA) containing 50 μM of caspase 3 (Ac-DEVD-AMC) substrate (Sigma-Aldrich, USA) at 37°C for 4 h. The level of fluorescence was determined using microplate reader (Infinite 200, Tecan, Switzerland; 360 nm excitation, 460 nm emission).

Caspase activity was analyzed as described previously (33 (link)). Isolated cells were washed twice with PBS, lysed (50 mM HEPES; 5 mM CHAPS; 5 mM dithiothreitol) (all Sigma-Aldrich) on ice for 20 min, and centrifuged (15 000 g for 15 min) at 4°C. Lavage fluid was diluted 1:1 with 2x concentrated lysing buffer. The proteins present in supernatants were quantified using Coomassie Protein Assay (Bio-Rad, California, USA) and diluted to the same concentration. 5 µg of protein samples were incubated in an assay buffer (20 mM HEPES; 2.5 mM CHAPS, 5 mM dithiothreitol, 2 mM EDTA) containing 50 µM of caspase 3 (Ac-DEVD-AMC) or caspase 8 substrate (Sigma-Aldrich) at 37°C for 4 h. The level of fluorescence was determined using a microplate reader (Infinite 200, Tecan, Switzerland; excitation – 360 nm for caspase 3 or 390 nm for caspase 8, emission - 460 nm for caspase 3 or 535 nm for caspase 8).