cd8α egfp plasmid  (Addgene inc)

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Development of a highly sensitive platform for protein-protein interaction detection and regulation of T cell function.

doi: 10.1073/pnas.2318190121

Figure Lengend Snippet: Fig. 5. IFNARRS compared with ELISpot assay. (A) Indicated numbers of Jurkat-CD8/anti-ESO-TCR cells were stimulated with 100 ng/mL PMA and 300 ng/mL calcium ionophore (PMA/Ca), with 100 ng/mL anti-CD3ε antibody, or medium only (−) for 13 h in a 96-well plate for ELISpot assay to measure the secreted IFN-γ from the T cells. The positive spots are visualized. (B) The numbers of positive spots in each well were determined and shown in logarithmic scale (mean ± SD of three wells). (C) Simultaneously, the same numbers of Jurkat-CD8/anti-ESO-TCR cells were added to the GRkS-974 cells seeded in a 96-well plate in advance (2 × 104 cells/well). After the same treatments as in A, Nluc assay was performed, taking the count of medium only with 1 × 101 Jurkat cells as 1 in the logarithmic scale (mean ± SD of four independent experiments), and shown. Asterisks indicate a significant difference from the control (medium only) (*P < 0.01, **P < 0.0001).

Article Snippet: The expression vectors for HER2 (#16257), CD8α- EGFP (#86051), TCRα/β/CD3ε/ζ (#89347), Cas9 (#52961), pSLCAR- CD1928z (#135991), and pSLCAR- CD19- BBz (#135992) were obtained from Addgene.

Techniques: Enzyme-linked Immunospot, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Development of a highly sensitive platform for protein-protein interaction detection and regulation of T cell function.

doi: 10.1073/pnas.2318190121

Figure Lengend Snippet: Fig. 6. Interactions of GRkS-974 cells and Jurkat cells were enhanced by the anti-CD3ε antibody, B7-1, and antigen peptide stimulation. (A) GRkS-974 cells (1 × 105 cells/well) were transfected with a B7-1-expressing vector or empty vector. Jurkat T cells (1 × 105 cells/well) stably expressing CD8α and anti-NY-ESO-1 TCR were added to cells 24 h after transfection and stimulated with 100 ng/mL anti-CD3ε antibody for 12 h. Nluc assay was performed by taking the empty vector transfection without anti-CD3ε antibody stimulation as 1 (mean ± SD of three independent experiments). (B) As indicated, GRkS-974 cells were transfected with vectors expressing B7-1 or secretory anti-CD3ε scFv-Sc. Jurkat cells stably expressing CD8α and anti-NY-ESO-1 TCR were added to the cells 24 h after transfection. After 12 h of coculture, Nluc assay was performed by taking the empty vector transfection as 1 (mean ± SD of three independent experiments). (C) GRkS-974 cells were transfected with the B7-1-expressing or empty vector. The cells were further incubated for 12 h with the indicated concentrations of NY-ESO-1 peptide to load on the surface MHC class I 24 h after transfection. After that, Jurkat cells stably expressing CD8α and anti-NY-ESO-1 TCR were added to the cells. After 12 h of coculture, Nluc assay was performed, taking the vector transfection without NY-ESO-1 peptide as 1 (mean ± SD of three independent experiments). Asterisks indicate a significant difference from the vector transfection without stimulation or between the indicated pairs (*P < 0.01, **P < 0.001). TCR, T cell receptor; and scFv-Sc, secretory single-chain variable fragment.

Article Snippet: The expression vectors for HER2 (#16257), CD8α- EGFP (#86051), TCRα/β/CD3ε/ζ (#89347), Cas9 (#52961), pSLCAR- CD1928z (#135991), and pSLCAR- CD19- BBz (#135992) were obtained from Addgene.

Techniques: Transfection, Expressing, Plasmid Preparation, Stable Transfection, Incubation