Journal: The Journal of Biological Chemistry

Article Title: Peptide-Major Histocompatibility Complex Dimensions Control Proximal Kinase-Phosphatase Balance during T Cell Activation

doi: 10.1074/jbc.M109.039966

Figure Lengend Snippet: TCR·CD3 triggering but not clustering is diminished by pMHC elongation. A , clean poly- l -lysine coated coverslips were incubated at 37 °C for 2 h with 50 μg/ml streptavidin/PBS. Biotinylated SCTs were immobilized on coverslips at comparable densities (Fig. S3 ). G10 cells were placed on SCT-coated coverslips and incubated for 1 min at 37 °C. The cells were fixed and permeabilized before serial staining CD3, Zap70, and CD45. The images were acquired using uniform settings between experiments. The cells adhered poorly on uncoated coverslips; therefore coverslips coated with biotinylated anti-HLA antibody was used as a control surface (without cognate TCR ligands). The scale bar represents 4 μm. B–D , the mean intensities for CD3, Zap70, and CD45 accumulation at the T cell interface was quantified. For a better appreciation of regions of TCR clustering pseudocolor scaled images of the CD3 distribution are shown (CD3 Scale) in A and E . The regions of CD3 clustering were arbitrarily defined by thresholding at two times the mean CD3 fluorescence intensity at the T cell interface (corresponding to the yellow regions in the pseudocolored scale images) and expressed as percentages of total CD3 interface fluorescence (Fig. S4 ). E , G10 cells were incubated with 10 μ m PP2 for 30 min prior to placing on to SCT-coated coverslips. Preparation and labeling were performed as described above. The scale bar represents 4 μm. The horizontal black bars represent the means. Statistical significance was determined by analysis of variance with correction for multiple comparisons. ns , p > 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: The samples were stained for CD3ε, Zap70, and CD45 by serial incubation of each antibody followed where appropriate by a species-specific and fluorophore-conjugated secondary antibody in the order mouse anti-CD3ε (UCHT1; AbD Serotec), anti-mouse Alexa 488 (Molecular Probes), rabbit anti-Zap70 (99F2; Cell Signaling Technology), anti-rabbit IgG Alexa 546 (Molecular Probes), and anti-human pan CD45 antibody directly conjugated with Alexa 647 (F10-89-4; AbD Serotec).

Techniques: Incubation, Staining, Control, Fluorescence, Labeling

Journal: The Journal of Biological Chemistry

Article Title: Peptide-Major Histocompatibility Complex Dimensions Control Proximal Kinase-Phosphatase Balance during T Cell Activation

doi: 10.1074/jbc.M109.039966

Figure Lengend Snippet: Small pMHC dimensions allow efficient segregation from CD45 following TCR engagement. A , experiments were performed as in . The data were acquired as 8-bit 1024 × 1024 pixel images. B , background subtracted images acquired in the CD3 and CD45 channels were analyzed for colocalization by determining Manders colocalization coefficients using ImageJ. The mean proportion of CD3 colocalized with CD45 expressed as a percentage (Manders M1 coefficient) was calculated from 20 interfaces chosen at random for each coated surface. Manders M2 coefficient (proportion of CD45 colocalized with CD3) is 100% for all. The horizontal black bars represent the means. Statistical significance was determined by analysis of variance with correction for multiple comparisons. *, p < 0.05; ***, p < 0.001. The scale bar represents 4 μm.

Article Snippet: The samples were stained for CD3ε, Zap70, and CD45 by serial incubation of each antibody followed where appropriate by a species-specific and fluorophore-conjugated secondary antibody in the order mouse anti-CD3ε (UCHT1; AbD Serotec), anti-mouse Alexa 488 (Molecular Probes), rabbit anti-Zap70 (99F2; Cell Signaling Technology), anti-rabbit IgG Alexa 546 (Molecular Probes), and anti-human pan CD45 antibody directly conjugated with Alexa 647 (F10-89-4; AbD Serotec).

Techniques:

Journal: iScience

Article Title: CX3CR1 deficiency-induced TIL tumor restriction as a novel addition for CAR-T design in solid malignancies

doi: 10.1016/j.isci.2023.106443

Figure Lengend Snippet: Key resources table

Article Snippet: Mouse anti-Human CD45 Antibody (Leukocyte marker) , Raybiotech , catalog number 188-10998-1.

Techniques: Marker, Recombinant, Enzyme-linked Immunosorbent Assay, Cell Isolation, Activation Assay, Staining, Over Expression, Construct, Plasmid Preparation, Software, Microscopy, Imaging, Flow Cytometry