Bca colorimetric assay

Cells were seeded into T-25 Flasks (2 × 10⁶ cells/flask) and the following day the cells were treated. After treatment, U266 and A2058 cells were washed with PBS and then A2058 cells were subjected to TrypLE treatment for dissociation. After subcultivation, the cells were centrifuged at 1,000 × g for 5 min. Then the supernates were transferred into clean tubes and the cells were rinsed with PBS and extracted with Lysis Buffer 17 (provided in the Human Apoptosis Array Kit). In order to separate also the apoptotic bodies induced by the treatments, the supernatants were centrifuged at 2,500 × g for 15 min at 4 °C, two times. Then the apoptotic-body containing pellet was extracted with Lysis Buffer 17. Before protein quantification, the lysed samples were mixed into each other and incubated with the lysis buffer for 20 min. Total protein quantity was evaluated by the colorimetric BCA assay (Thermo Scientific, Waltham, MA USA). Protein extracts (225 μg) were used for apoptosis array analysis.

Lysates from C2C12 myotubes, soleus, and triceps were obtained by homogenization in co-IP buffer (10% NP-40, 20% 20 mM NaF, 1% Triton X-100) supplemented with complete protease inhibitor tablet (Roche Applied Science) and 1 mM leupeptin and 1 mM pepstatin A (MilliporeSigma). Cells were collected and lysed at 4°C for 30 minutes. After centrifugation (16,000g, 4°C, 20 minutes), the soluble fractions were collected, and the concentration was measured using a colorimetric BCA assay (23225; Thermo Fisher Scientific). Soluble homogenates were precleared with Dynabead Protein G beads (Thermo Fisher Scientific) for 1 hour, and supernatants were incubated with the specific antibodies directed against the protein of interest at 4°C for 12 to 24 hours. Dynabead Protein G beads were then added for 2 hours to capture the immune complex. Beads were washed 3 times with co-IP buffer supplemented with 0.1% CHAPS. For all experiments, 2 negative controls consisted of a sample lacking the primary antibody and a sample incubated with another primary antibody from the same serotype as the antibody of interest. Resulting beads were eluted with Laemmli buffer and subjected to SDS-PAGE followed by immunoblot.

The total cell count was determined for a fresh fluid specimen using slides with counting grids (Hycor biomedical, Indianapolis, IN, USA). The cell pellet was diluted in saline, and differential cell counts were performed on cytospin preparations (Cytospin 3; Shandon Scientific, Cheshire, UK) stained with Diff-Quick stain (Baxter Diagnostics, McGaw Park, IL, USA). Bronchoalveolar lavage fluid was centrifuged (1500 rpm, 13 min at 4 °C), and cell-free supernatants were stored at −80 °C for subsequent assessment of protein content (using a colorimetric BCA assay, Thermo Scientific, Rockford, IL, USA).