plate coated okt3  (Miltenyi Biotec)

Journal: Oncoimmunology

Article Title: A T-cell engaging bispecific antibody with a tumor-selective bivalent folate receptor alpha binding arm for the treatment of ovarian cancer

doi: 10.1080/2162402X.2022.2113697

Figure Lengend Snippet: TNB-928B is a fully human bispecific antibody engaging FRα and CD3. (a) TNB-928B was constructed using knobs-into-holes technology. The schematic shows the format of the antibody. (b) Cell surface affinity of TNB-928B to FRα expressed on IGROV-1 cells was determined by Scatchard analysis. (c) Co-culture cytotoxicity assay with T-cells (E:T ratio of 10:1) showing cell lysis of IGROV-1 tumor cells after 48 h. Monovalent and bivalent FRαxCD3_OKT3 bispecific molecules are the positive controls for monovalent FRαxCD3_F2B and TNB-928B respectively.

Article Snippet: Prior to injection, PBMCs were activated for 3 d with 1 µg/mL plate-coated anti-CD3 (eBioscience, clone OKT3) and 1 µg/mL soluble anti-CD28 (eBioscience, clone CD28.2).

Techniques: Construct, Co-Culture Assay, Cytotoxicity Assay, Lysis

Journal: Journal for Immunotherapy of Cancer

Article Title: Preclinical characterization of the novel anti-SIRPα antibody BR105 that targets the myeloid immune checkpoint

doi: 10.1136/jitc-2021-004054

Figure Lengend Snippet: BR105 does not inhibit T cell activation and proliferation. (A) Binding of indicated mAbs to SIRPγ was determined by ELISA. Data represent mean±SEM; representative of n=3 is shown. (B) PBMCs were cultured with 10 µg/mL of indicated mAbs in OKT3-coated plates for 3 days. Proliferation measured by CellTiter-Glo was normalized to control (PBMCs without addition of mAbs). (C) Dendritic cells and allogeneic PBMCs from three different donors were cultured 5 days with indicated mAbs at 10 µg/mL. Interferon γ secretion was quantified by ELISA and normalized to control (Dendritic cells and PBMCs without addition of mAbs). Data represent mean ± SEM; representative of n=3 donor is shown. *Indicate statistical differences compared with the respective isotype control group: *p<0.05, ***p<0.001, ****p<0.0001. ns, not significant.

Article Snippet: 96-well plates were coated with 0.1 μg/mL OKT3 (ACROBiosystems) at 4°C overnight.

Techniques: Activation Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Cell Culture, Control

Journal: bioRxiv

Article Title: Mutation of an L-Type Calcium Channel Gene Leads to a Novel Human Primary Cellular Immunodeficiency

doi: 10.1101/864280

Figure Lengend Snippet: (A) Cav1.4 gene expression by qRT-PCR in Jurkat cells after 48 hours of Cav1.4 shRNA transfection. (B) Jurkat T cells, transfected with either Cav1.4 shRNA or scrambled control shRNA were loaded with Ca 2+ dyes and analyzed by flow cytometry. After 30 seconds the cells were activated with α-CD3. Knockdown was performed once, Ca 2+ flux experiment is a representative of two technical replicates.

Article Snippet: Cells were stimulated with α-CD3 (coated plates, OKT3) and α-CD28/CD49d (soluble, L293, L25, BD Biosciences) for 4.5 hours, in the presence of brefeldin A (BD Biosciences) and monensin (BD Biosciences).

Techniques: Expressing, Quantitative RT-PCR, shRNA, Transfection, Flow Cytometry

Journal: bioRxiv

Article Title: Mutation of an L-Type Calcium Channel Gene Leads to a Novel Human Primary Cellular Immunodeficiency

doi: 10.1101/864280

Figure Lengend Snippet: PBMCs of patients (n=3) and healthy donors (n=4) were stimulated with α-CD3 and α-CD28/49d for 4.5 hours and subsequently stained with various antibodies for extracellular markers and intracellular cytokines. The frequencies of CD38+, IFNγ+, and IL-2+ lymphocytes are shown for CD4 and CD8 non-naïve T lymphocytes, which are classified as NOT CD27+ CD45RA+. This experiment was only done once.

Article Snippet: Cells were stimulated with α-CD3 (coated plates, OKT3) and α-CD28/CD49d (soluble, L293, L25, BD Biosciences) for 4.5 hours, in the presence of brefeldin A (BD Biosciences) and monensin (BD Biosciences).

Techniques: Staining