intestinal epithelial cell line ncm460  (Procell Inc)

Journal: Journal of Immunology Research

Article Title: The Generation and Characterization of Recombinant Protein and Antibodies of Clostridium perfringens Beta2 Toxin

doi: 10.1155/2016/5708468

Figure Lengend Snippet: The cytotoxicity of rCPB2 toxin in human colon NCM460 cells. The cytotoxicity of purified rCPB2 protein was ascertained by determining its concentration causing 50% cell death (cytotoxicity 50, CT50) in a given time point in terms of an MTT assay. (a) A representative image (×400) of control NCM460 showed normal morphology of cells. (b) A representative image of NCM460 cells treated with rCPB2 at 20 μ g/mL for 12 h exhibited a morphological change of cell death. (c) A representative image of NCM460 was treated for 12 h with supernatant of C. perfringens C59-44 strain. (d) The viability of NCM460 cells treated with indicated concentrations of rCPB2 for indicated times determined by an MTT assay. The data was expressed as the mean ± SD of fold changes of viable cells against the control untreated cells from three triplicated independent experiments. Bars in (a) and (b): 50 μ m.

Article Snippet: The intestinal epithelial cell line NCM460 was procured from the National Centre for Cell Science (Shanghai, China).

Techniques: Purification, Concentration Assay, MTT Assay, Control

Journal: Journal of Immunology Research

Article Title: The Generation and Characterization of Recombinant Protein and Antibodies of Clostridium perfringens Beta2 Toxin

doi: 10.1155/2016/5708468

Figure Lengend Snippet: rCPB2 toxin induces apoptosis in NCM460 cells. The NCM460 cells were treated with indicated concentrations of rCPB2, and the cell death was analyzed by a cytometric assay. (a) Representative dot plots of three independent experiments of flow cytometry analysis showed an increased fraction of apoptotic NCM460 cells treated with indicated concentrations of rCPB2 for 12 h. (b) The quantitative analysis showed a dose-dependent cell apoptosis induced by rCPB2; the rCPB2 exhibited an ability to induce cell apoptotic death rather than necrosis. (c) Immunoblotting assay of proapoptotic protein caspase-3 and Bax showed potential of rCPB2-induced NCM460 cells. Data represented the mean ± SD from three independent experiments in (b) compared to a naive control, ∗ p < 0.05; ∗∗ p < 0.01.

Article Snippet: The intestinal epithelial cell line NCM460 was procured from the National Centre for Cell Science (Shanghai, China).

Techniques: Flow Cytometry, Western Blot, Control

Journal: Journal of Immunology Research

Article Title: The Generation and Characterization of Recombinant Protein and Antibodies of Clostridium perfringens Beta2 Toxin

doi: 10.1155/2016/5708468

Figure Lengend Snippet: Ability of rCPB2 binding to NCM460 cells characterized by an immunofluorescent assay. NCM460 cells were incubated with rCBP2 at concentration of 15 μ g/mL for indicated times. The cells were then used for accessing rCPB2 protein and cell membrane with McAb 1E23 to CPB2 (1 : 100) and rabbit polyclonal antibody to Annexin A2 (1 : 200), respectively. FITC-labeled donkey anti-mouse IgG (1 : 500) and Rhodamine- (TRITC-) conjugated goat anti-rabbit IgG (1 : 250) were used as secondary antibodies for visualizing respective proteins of interest. DAPI was used for nuclear staining. (a–f) Representative images of immunofluorescent staining for cells incubated with rCPB2 at indicated times of 5 min (a), 30 min (b), 2 h (c), 4 h (d), 6 h (e), and 12 h (f). The results showed that rCPB2 was able to bind to the membrane of NCM460 cell and translocate into the cytoplasm of cells. Insets were the enlarged boxed regions of respective images in (a–f). Bars in (a–f): 50 μ m.

Article Snippet: The intestinal epithelial cell line NCM460 was procured from the National Centre for Cell Science (Shanghai, China).

Techniques: Binding Assay, Incubation, Concentration Assay, Membrane, Labeling, Staining

Journal: Journal of Immunology Research

Article Title: The Generation and Characterization of Recombinant Protein and Antibodies of Clostridium perfringens Beta2 Toxin

doi: 10.1155/2016/5708468

Figure Lengend Snippet: Characterizations of McAbs to CPB2 by an ELISA and abilities to neutralize rCPB2-induced cytotoxicity in vitro . (a) Titration of ascites of McAbs to CPB2 determined by an ELISA. An ELISA plate was coated with rCPB2 at concentration of 3.0 μ g/mL; ascites of McAbs were diluted by 2-fold series dilution and incubated with above coated antigens; a reaction of antigen-antibody was detected by an ELISA. The result indicated that McAbs 1E23, 2G7, and 2H7 could be used for detecting CPB by an ELISA. (b) A capacity of McAbs to neutralize the toxicity of rCPB2 toxin in vitro . A 2x LD50 (30 μ g/mL) of rCPB2 toxin was mixed with 200 μ L of 50x diluted McAbs and incubated at 37°C for 2 h; the mixture was then applied to NCM460 cells incubated at 37°C for additional 12 h. The cell viability was measured by an MTT assay. The result showed abilities of tested McAbs to neutralize the cytotoxicity of rCPB2 in vitro . Among these McAbs, 1E23 displayed the most potential to neutralize the cytotoxicity of rCPB at a dose of 30 μ g/mL.

Article Snippet: The intestinal epithelial cell line NCM460 was procured from the National Centre for Cell Science (Shanghai, China).

Techniques: Enzyme-linked Immunosorbent Assay, In Vitro, Titration, Concentration Assay, Incubation, MTT Assay