HepG2 with a density of 1 × 106 cells/mL was counted and 2 mL/well was added into the six-well plate. After being cultured for 24 h, Na[OsVI(N)(tpm)2] at the indicated concentrations was added and reacted for 24 h. Cells were scraped with a scraper, collected, and washed twice with cold PBS. Cells were lysed with a cell lysis buffer, of which the main active component is 1% Triton X-100, with protease inhibitor phenylmethanesulfonyl fluoride (PMSF). Protein quantifications were measured using the BCA Protein Assay Kit (Beyotime) by Infinite M200 (Männedorf, Swiss, Tecan). Equal amounts of cellular proteins were mixed with SDS-PAGE Sample Loading Buffer, 5X (Beyotime), boiled at 95 °C for 5 min, and run on 12–15% separation gel. Protein was transferred to the nitrocellulose membrane (BOSTER). The membrane was blocked with 5% nonfat milk in TBST (1X, 0.1% Tween-20) for 40 min at RT, washed with TBST for 5 s, and then probed with primary antibody at 4 °C overnight followed by secondary antibody. Next, the ECL Plus detection kit (Beyotime) was added and the membrane was visualized using High ChemiDoc XRS (Bio-Rad ChemiDoc XRS+, Hercules, CA, USA).
High chemidoc xrs
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The High ChemiDoc XRS is a compact and versatile imaging system designed for the detection and analysis of chemiluminescent, colorimetric, and fluorescent signals. It provides high-quality image capture and analysis capabilities for a range of life science applications.
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2 protocols using high chemidoc xrs
1
Evaluating Osmium-Induced Cellular Responses
2
Mitochondrial Membrane Potential Assay in NCI-H460 Cells
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NCI-H460 cells were cultured with complex 6 for 24 h at indicated concentrations. The treated cells were digested and collected. Then, the cells were incubated with JC-1 staining buffer for 20 min at 37 °C in the dark. The cells were washed with JC-1 washing buffer (1×) twice and analyzed by flow cytometry to detect the red/green fluorescence intensity.
Western blot analysis NCI-H460 cells were seeded in 6-well plates. After treatment with 6 at different concentrations and times, the cells were lysed in cell lysis buffer, which contains a protease inhibitor. The protein was extracted and quantified using the BCA Protein Assay Kit. The protein samples were separated on 8-15% SDS-PAGE and transferred onto a nitrocellulose membrane (BOSTER). After blocking with 5% nonfat milk for 1 h at room temperature, the membranes were probed with the primary antibody overnight at 4 °C and incubated with a secondary antibody (anti-rabbit or anti-mouse, 1 : 10 000) for 1 h at room temperature. The target proteins were examined using a High ChemiDoc XRS (Bio-Rad ChemiDoc XRS+, USA).
Western blot analysis NCI-H460 cells were seeded in 6-well plates. After treatment with 6 at different concentrations and times, the cells were lysed in cell lysis buffer, which contains a protease inhibitor. The protein was extracted and quantified using the BCA Protein Assay Kit. The protein samples were separated on 8-15% SDS-PAGE and transferred onto a nitrocellulose membrane (BOSTER). After blocking with 5% nonfat milk for 1 h at room temperature, the membranes were probed with the primary antibody overnight at 4 °C and incubated with a secondary antibody (anti-rabbit or anti-mouse, 1 : 10 000) for 1 h at room temperature. The target proteins were examined using a High ChemiDoc XRS (Bio-Rad ChemiDoc XRS+, USA).
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