celltrace violet cell proliferation dye  (Thermo Fisher)

Journal: Molecular Cancer Therapeutics

Article Title: Development of a Novel Bifunctional Anti-CD47 Fusion Protein with Improved Efficacy and a Favorable Safety Profile

doi: 10.1158/1535-7163.MCT-24-0917

Figure Lengend Snippet: CO-001 induces cancer cell phagocytosis via blocking the CD47 interaction with SIRPα and mediates direct PCCD. A, Jurkat cells (5 × 10 5 cells/mL) were incubated with 100 μg/mL human IgG4 isotype control, CO-001, B6H12, or 2D3, followed by incubation with 10 μg/mL FITC-conjugated SIRPα. B, CellTrace-labeled Jurkat cells co-cultured with DiO-labeled RAW264.7 macrophages and treated with CO-001, anti-CD47 sequence analogues AO-176 or magrolimab, benchmark anti-CD47 B6H12, or human IgG4 isotype control for 2 hours. C, Time-lapse imaging of Jurkat cells treated with the human IgG4 isotype control or CO-001 (1 μg/mL) at the indicated time points (H = hours) using the IncuCyte Live Cell Imaging System. Scale bar, 200 μm. D, Representative FACS plots showing Jurkat cells treated with 1 μg/mL the human IgG4 isotype control or CO-001 for 3 hours and then stained with annexin V and 7-AAD. The gated population, indicated by a red rectangle, indicates the population of the cells that is undergoing PCCD with 11% undergoing PCCD for IgG4-treated cells and 58% for CO-001 treated. E, Jurkat cells (5 × 10 5 cells/mL) were treated with CO-001 at the indicated concentrations in μg/mL or human IgG4 isotype control (1 μg/mL) for 0.5, 1, or 3 hours, followed by annexin V and 7-AAD staining. F, Reh cells (5 × 10 5 cells/mL) were treated with CO-001 or AO-176 at the indicated concentrations in μg/mL or human IgG4 isotype control (10 μg/mL) for 3 hours, followed by annexin V and 7-AAD staining. A , B , and E, Data presented as the mean ± SD. Significance was determined by ANOVA analysis *, P < 0.01 relative to isotype control, §, P < 0.01, relative to the sample treated with CO-001 at the same concentration, n = 3 to 5. G, Binding of CO-001 to various cell lines derived from hematologic malignancies (Jurkat, MOLT-4, CCRF-CEM, and KG-1a) and RBCs relative to the human IgG4 isotype control. The EC 50 was determined using four-parameter curve fitting analysis in GraphPad Prism. H, Hemagglutination assay of freshly isolated RBCs (2% v/v in PBS) incubated with increasing concentrations of the CO-001, sequence analogues of AO-176 and magrolimab, or IgG4 isotype control. A small punctate circle indicates no hemagglutination, whereas a diffuse hazy pattern indicates hemagglutination. MFI, mean fluorescence intensity.

Article Snippet: Target cancer cells were collected by centrifugation and stained with 1 μL/mL CellTrace Violet Cell Proliferation Dye according to the manufacturer’s protocol (Thermo Fisher Scientific, cat # C34557 ).

Techniques: Blocking Assay, Incubation, Control, Labeling, Cell Culture, Sequencing, Analogues, Imaging, Live Cell Imaging, Staining, Concentration Assay, Binding Assay, Derivative Assay, Hemagglutination Assay, Isolation, Fluorescence

Journal: Molecular Cancer Therapeutics

Article Title: Development of a Novel Bifunctional Anti-CD47 Fusion Protein with Improved Efficacy and a Favorable Safety Profile

doi: 10.1158/1535-7163.MCT-24-0917

Figure Lengend Snippet: Development of the CO-005 drug candidate with an improved RBC safety profile. A, Cartoon representation of the structure of the CO candidates CO-001, CO-004, and CO-005. B, Incubation of freshly isolated RBCs (2% v/v in PBS) with increasing concentrations of CO-001, CO-004, CO-005, or human IgG4 isotype control. A small punctate circle indicates no hemagglutination, whereas a diffuse hazy pattern indicates hemagglutination. C, Binding of CO-001, CO-005, CO-004, and human IgG4 isotype control to RBCs derived from a healthy human donor. D, Jurkat cells (5 × 10 5 cells/mL) were treated with CO-001, CO-004, or CO-005 at the indicated concentrations in μg/mL or human IgG4 isotype control (1 μg/mL) for 3 hours, followed by annexin V and 7-AAD staining. E, Phagocytosis activity of CO-001 fusion proteins in Jurkat cells using DiO-labeled RAW264.7 macrophages at the indicated concentrations for 2 hours. Phagocytosed target cells were analyzed by flow cytometry as %CellTrace + DiO + cells. D and E, Data are presented as the mean ± SD. Significance was determined by ANOVA analysis *, P < 0.01 relative to isotype control, §, P < 0.01, relative to the sample treated with CO-001 at the same concentration, n = 3 to 5. MFI, mean fluorescence intensity.

Article Snippet: Target cancer cells were collected by centrifugation and stained with 1 μL/mL CellTrace Violet Cell Proliferation Dye according to the manufacturer’s protocol (Thermo Fisher Scientific, cat # C34557 ).

Techniques: Incubation, Isolation, Control, Binding Assay, Derivative Assay, Staining, Activity Assay, Labeling, Flow Cytometry, Concentration Assay, Fluorescence