Cy3 conjugated goat anti mouse igg

Briefly, RAW264.7 macrophages were cultured in DMEM culture medium supplemented with 10% FBS in a humidified atmosphere of 5% CO₂ at 37 °C. After the cells grew into a monolayer, the cells were transferred to a 6-well microplate (10⁵ cells/mL), and then rGSTO2 (20 μg/mL) or P28a protein and PBS (control group) were added to each well and co-incubated with the cells for 2 h of 5% CO₂ at 37 °C. Cells were fixed with 4% paraformaldehyde at ambient temperature for 15 min, washed three times with PBS (5 min each time) and then blocked with 4% BSA in PBS at 37 °C for 1 h. Cells were incubated with mouse anti-rGSTO2 IgG, anti-P28a IgG and naïve serum (1:500 dilution) at 4 °C overnight, respectively followed by staining with Cy3-conjugated goat anti-mouse IgG (1:500, Abcam, UK) at 37 °C for 1 h. Then, the nuclei of cells were stained by 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI, Beyotime, Shanghai, China) for 5 min. Finally, these cells were visualized using a confocal microscopy (Leica Microsystems GmbH, Wetzlar, Germany).

HCT-8 cells (2.5 × 10⁵ per well) were seeded in the ultralow attachment 6-well plates (Corning Incorporated, Corning, NY, USA). Spheroid cells were cultivated for 6 days in the spheroid culture media (DMEM containing B27 (Life Technology, Waltham, MA, USA)), 20 ng/ml of bFGF (PEPROTECH, Rocky Hill, NJ, USA), 20 ng/ml of recombinant human EGF (Cell Signaling), and 1% antibiotics. To verify the competence of colonospheres, 6-day cultured spheroids were fixed with 4% paraformaldehyde containing 1% triton X-100 at 4 °C overnight, then fixed with 4% paraformaldehyde over 2 h. Spheroids were washed with PBS, blocked with 3% BSA, and then incubated with mouse anti-human CD44 (1:200, Cell signaling) at 4 °C overnight. The fixed spheroids were subsequently washed in PBS, incubated with Cy3-conjugated goat anti-mouse IgG (Abcam, Cambridge, UK) for 2 h at room temperature, washed in PBS, and counterstained with 100 ng/ml DAPI (absorbance at 405 nm, Sigma-Aldrich) in PBS for 10 min. Spheroids were washing with PBS 5 times for 8 min each, observed, and analyzed under a confocal laser scanning microscope (FV1000, Olympus, Nagano, Japan). To compare sphere sizes, about 200 spheroids per group were randomly selected, after which their perimeters were measured and statistically analyzed.

Rabbit anti-phosphorylated and anti-total p38MAPK mAb, rabbit anti-phosphorylated and anti-total IκB mAb, rabbit anti-phosphorylated and anti-total AKT mAb, rabbit anti-Mcl-1 mAb, rabbit anti-procaspase 3 mAb, mouse anti-active caspase-3 mAb, mouse anti-β-actin mAb, rabbit anti-IgG, and mouse anti-IgG were purchased from Cell Signaling Technology (Beverly, MA, USA). Mouse anti-His mAb was purchased from Affinity Biosciences, Inc (OH, USA). The rabbit anti-C. psittaci mAb was a gift from Professor G. Zhong (University of Texas Health Science Center at San Antonio, TX). The rabbit anti-SOD2 antibody, mouse anti-Bax antibody was purchased from ImmunoWay Biotechnology Company (Plano, TX, USA). The inhibitors SB203580, BAY117082, LY294002, staurosporine (STS), and Cy2-conjugated goat anti-rabbit IgG, DAPI, Cy3-conjugated goat anti-mouse IgG, and Cy3-conjugated goat anti-rabbit IgG and mouse anti-NF-κB p65 antibody were obtained from Abcam (Cambridge, MA, USA), recombinant human IL-8 from (Biolegend, CA, USA), anti-IL-8 mAb was obtained from GeneTex, USA.