Journal: Molecular and Cellular Biochemistry

Article Title: A fully human anti-c-Kit monoclonal antibody 2G4 inhibits proliferation and degranulation of human mast cells

doi: 10.1007/s11010-022-04557-3

Figure Lengend Snippet: 2G4 and 4C9 antibodies bind to c-Kit and inhibit c-Kit activation in LAD2 cells. A LAD2 cells were incubated with 2G4, 4C9, or normal human IgG1 at the indicated concentrations for 1 h. After washing, FITC-conjugated secondary antibody was added for 1 h. The fluorescence was detected by flow cytometry. B , C SCF-starved LAD2 cells were incubated with 2G4 antibody ( B ) or 4C9 antibody ( C ) at the indicated concentration for 1 h. Thereafter, the cells were stimulated by 100 ng/mL of SCF for an additional 10 min. Phosphorylation of c-Kit, Akt, and Erk1/2 was analyzed by Western blotting. α-Tubulin was used as a loading control

Article Snippet: The cells (2 × 10 5 cells) were stained with 2G4, 4C9, or normal human IgG1 (Sino Biological, Beijing, China) at the indicated concentrations at 4 °C for 1 h. The cells were then rinsed thrice in PBS containing 2% BSA and stained with goat anti-human IgG secondary antibody (0.3 μg/mL, Invitrogen, CA, USA) at 4 °C for 1 h. After washing, the fluorescence signal was detected using CyFlow Cube6 (Sysmex Partec, Goerlitz, Germany), and the data analysis was performed using FCS Express 6 Flow (De Novo software, CA, USA).

Techniques: Activation Assay, Incubation, Fluorescence, Flow Cytometry, Concentration Assay, Western Blot, Control

Journal: Molecular and Cellular Biochemistry

Article Title: A fully human anti-c-Kit monoclonal antibody 2G4 inhibits proliferation and degranulation of human mast cells

doi: 10.1007/s11010-022-04557-3

Figure Lengend Snippet: 2G4 antibody inhibits cell proliferation and migration in LAD2 cells. A , B LAD2 cells were incubated with 2G4, 4C9, or normal human IgG1 at the indicated concentrations in culture medium with SCF ( A ) or without SCF ( B ) for 7 days. Thereafter, cells were stained with 10 μM Hoechst 33342 and counted using a Celigo Imaging Cytometer. The black dashed line (100%) indicates normalized cell counts in the well without SCF and antibodies at 7 days. C Migration assay was carried out in a 6-transwell plate with 8 μm pores. LAD2 cells (1 × 10 6 cells) and the antibodies (1 μg/mL) were added into the upper chamber and SCF (100 ng/mL) was added into the lower chamber for 24 h. Migrated cells in the lower chamber were microscopically counted using a HPF in five different fields. All results represent the mean ± SD of three independent experiments. * vs. SCF − /Antibody − and # vs. SCF + /Antibody − . * P < 0.05, and # P < 0.05 (Student’s two-tailed t test)

Article Snippet: The cells (2 × 10 5 cells) were stained with 2G4, 4C9, or normal human IgG1 (Sino Biological, Beijing, China) at the indicated concentrations at 4 °C for 1 h. The cells were then rinsed thrice in PBS containing 2% BSA and stained with goat anti-human IgG secondary antibody (0.3 μg/mL, Invitrogen, CA, USA) at 4 °C for 1 h. After washing, the fluorescence signal was detected using CyFlow Cube6 (Sysmex Partec, Goerlitz, Germany), and the data analysis was performed using FCS Express 6 Flow (De Novo software, CA, USA).

Techniques: Migration, Incubation, Staining, Imaging, Cytometry, Two Tailed Test

Journal: Molecular and Cellular Biochemistry

Article Title: A fully human anti-c-Kit monoclonal antibody 2G4 inhibits proliferation and degranulation of human mast cells

doi: 10.1007/s11010-022-04557-3

Figure Lengend Snippet: 2G4 antibody inhibits IgE-mediated degranulation enhanced by SCF. A LAD2 cells were SCF-starved for 24 h. The cells were then incubated with 2G4, 4C9, normal human IgG1, or streptavidin for 1 h, and β-hexosaminidase release assay was performed. B , C LAD2 cells were sensitized with biotinylated human IgE ( B ) or IFN-γ ( C ) for 24 h in SCF-deficient medium. The cells were then incubated with 2G4, 4C9, normal human IgG1 or streptavidin for 1 h. Following this, β-hexosaminidase release assay was carried out. Streptavidin was used as a positive control to crosslink biotinylated-IgE. D LAD2 cells were SCF-starved and sensitized with biotinylated human IgE for 24 h. Cells were treated with antibodies (2G4, 4C9, or normal human IgG1), SCF (100 ng/mL), and streptavidin (2 ng/mL) in sequence at 30 min intervals. After 30 min of streptavidin treatment, β-hexosaminidase release assay was performed to analyze the degranulation of LAD2. All results represent the mean ± SD of three independent experiments. *, **, and *** vs Untreated, # vs. SCF − /Streptavidin − , § vs. SCF + /Streptavidin − , † vs. SCF − /Streptavidin + , and ‡ vs. SCF + /Streptavidin + . * P < 0.05, ** P < 0.01, *** P < 0.001, # P < 0.05, ## P < 0.01, ### P < 0.001, § P < 0.05, §§ P < 0.01, §§§ P < 0.001, † P < 0.05, †† P < 0.01, ††† P < 0.001, ‡ P < 0.05, ‡‡ P < 0.01, and ‡‡‡ P < 0.001 (one-way ANOVA with Dunnett’s post-test)

Article Snippet: The cells (2 × 10 5 cells) were stained with 2G4, 4C9, or normal human IgG1 (Sino Biological, Beijing, China) at the indicated concentrations at 4 °C for 1 h. The cells were then rinsed thrice in PBS containing 2% BSA and stained with goat anti-human IgG secondary antibody (0.3 μg/mL, Invitrogen, CA, USA) at 4 °C for 1 h. After washing, the fluorescence signal was detected using CyFlow Cube6 (Sysmex Partec, Goerlitz, Germany), and the data analysis was performed using FCS Express 6 Flow (De Novo software, CA, USA).

Techniques: Incubation, Release Assay, Positive Control, Sequencing

Journal: BioMed Research International

Article Title: Cytotoxic and Apoptotic Effects of Govaniadine Isolated from Corydalis govaniana Wall. Roots on Human Breast Cancer (MCF-7) Cells

doi: 10.1155/2018/3171348

Figure Lengend Snippet: Cell apoptosis observed using fluorescence microscope (200×). Cells were treated with different dose of govaniadine for 24 h and stained with (row (a)) acridine orange-ethidium bromide and (row (b)) Hoechst 33258. (A and A′) untreated cells (0.1% DMSO), (B and B′) cells treated with 2 μ M of govaniadine, (C and C′) cells treated with 4 μ M of govaniadine, (D and D′) cells treated with 8 μ M of govaniadine, and (E and E′) cells treated with 16 μ M of govaniadine. Scale bar: 100 μ m.

Article Snippet: After 24 h, apoptosis mediated by the govaniadine on MCF-7 cells was detected by the Annexin V/ Propidium Iodide (PI) apoptosis detection kit (Santa Cruz, Texas, USA) according to the manufacturer's instructions by using a Partec Cyflow® Cube 6 Flow Cytometer.

Techniques: Fluorescence, Microscopy, Staining

Journal: BioMed Research International

Article Title: Cytotoxic and Apoptotic Effects of Govaniadine Isolated from Corydalis govaniana Wall. Roots on Human Breast Cancer (MCF-7) Cells

doi: 10.1155/2018/3171348

Figure Lengend Snippet: Induction of apoptosis in MCF-7 cells by govaniadine determined by Annexin V-PI flow cytometry technique. (a) Control (0.1% DMSO), (b), (c), and (d) treated with govaniadine 4, 8, and 16 μ M, respectively. The lower left quadrant represents intact viable cells (Annexin-FITC and PI negative). The upper left quadrant represents early apoptotic cells (Annexin-FITC positive and PI negative). The upper right region represents late apoptotic cells (Annexin-FITC negative and PI positive). The lower left quadrant represents necrotic cells (Annexin-FITC and PI positive). The data are presented as dot plots of Annexin V/FITC against PI of at least three independent tests. Data were analyzed by specific software, FCS Express 5.

Article Snippet: After 24 h, apoptosis mediated by the govaniadine on MCF-7 cells was detected by the Annexin V/ Propidium Iodide (PI) apoptosis detection kit (Santa Cruz, Texas, USA) according to the manufacturer's instructions by using a Partec Cyflow® Cube 6 Flow Cytometer.

Techniques: Flow Cytometry, Control, Software