Journal: bioRxiv

Article Title: CARs are organized in nanodomains in the plasma membrane of T cells that accumulate at tumor contact sites

doi: 10.1101/2023.07.19.549702

Figure Lengend Snippet: a, Immunological synapse formation between a representative CD4 + T cell expressing CD19-specific CARs and ZAP70 labeled with GFP (rainbow LUT), and a Raji cell (magenta) stained for CD20 by antibodies conjugated to ATTO643. b, A second example of synapse formation between a CD4 + CD19-specific CAR-T cell and a Raji cell. An increase in fluorescence signal of GFP channel (rainbow LUT) can be seen at the cell-cell junction confirming ZAP70 accumulation at the cell-cell contact site. c , 3D volume rendering of a CD4 + CD19-specific CAR-T cell and a Raji cell forming an immunological synapse. Scattered accumulation of GFP signal (white pointers) can be visualized at the contact site. d, Immunological synapse formation between a representative CD4 + T cell (rainbow color scale) without CAR construct expressing ZAP70-GFP and a Raji cell (magenta). Synapse formation was induced by addition of staphylococcal enterotoxin type E (SEE). e, No immunological synapse formation is seen between CD4 + T cells without CAR construct expressing ZAP70-GFP and Raji cells. f, Side view of a CD4 + T cell without CD19-CAR at the site of synapse formation (induced by addition of SEE) shows accumulation of ZAP70-GFP signal resembling the classical ‘bull’s eye’ organization. g, Scattered clustering of ZAP70-GFP at multiple foci (indicated with white arrows) interleaved by lower fluorescence intensity regions unlike the classical organization can be seen at the site of synapse formation for a CD4 + CD19-specific CAR-T cell (right panel). Scale bars, 5 µm.

Article Snippet: Prior to functional testing, EGFRt-positive T cells were enriched using biotin-conjugated anti-EGFR mAb and anti-biotin beads (Miltenyi Biotec), and subsequently expanded using a rapid expansion protocol based on CD3-TCR-activation via OKT-3 (Miltenyi Biotec) and irradiated donor-mismatched PBMCs (all CAR-T cells, unless indicated) or irradiated feeder cells expressing CD19 or anti-CAR surface-proteins (LV/SB comparison of CD19 and ROR2 CAR-T cells) for 7-14 days.

Techniques: Expressing, Labeling, Staining, Fluorescence, Construct

Journal: PLoS ONE

Article Title: Bridging the Gap between Single Molecule and Ensemble Methods for Measuring Lateral Dynamics in the Plasma Membrane

doi: 10.1371/journal.pone.0078096

Figure Lengend Snippet: Differential interference contrast images (left column, scale bar = 10 µm) and superimposed trajectories from SPT analysis (right column, scale bar = 1 µm) in selected regions-of-interests (ROIs). (a) Four color single QD time-lapse imaging of an artificial lipid, biotin-cap-DPPE, labeled by a four color combination of sAv-QD705 (red), sAv-QD655 (green), sAv-QD605 (blue), and sAv-QD565 (yellow). (b) Three color single orthogonal QD imaging of the sphingolipid, G M1 , with ChToxB-QD705 conjugates (red), ACP-CD59with CoA-QD655 conjugates (green), and BLAP-EGFR with sAv-QD605 conjugates (blue).

Article Snippet: In brief, expression of the fusion proteins biotin ligase acceptor peptide- epidermal growth factor receptor (BLAP-EGFR) and acyl carrier protein - glucosylphosphatidylinositol anchored protein (ACP-CD59)(New England Biolabs, Ipswich, MA) in MEF cells was done by transient co-transfection of four DNA plasmids, BirA-KDEL (1 µg), BLAP-EGFR (1 µg), ACP-CD59 (1 µg), and a plasma membrane fluorescent protein marker, K-Ras2-YFP (0.25 µg, ATCC plasmid 10089283) per 25,000 cells.

Techniques: Imaging, Labeling

Journal: PLoS ONE

Article Title: Bridging the Gap between Single Molecule and Ensemble Methods for Measuring Lateral Dynamics in the Plasma Membrane

doi: 10.1371/journal.pone.0078096

Figure Lengend Snippet: The slopes of each average correlation function ln (r( k ,τ))from two ROIs at different τ values are plotted as a function of τ. The slope of each plot gives the diffusion coefficients listed in and . (a) Four color single QD time-lapse imaging of an artificial lipid, biotin-cap-DPPE, labeled by a four color combination of sAv-QDs. (b) Three color single orthogonal QD imaging of the sphingolipid, G M1 , with ChToxB-QD705s, ACP-CD59 with CoA-QD655s, and BLAP-EGFR with sAv-QD605s.

Article Snippet: In brief, expression of the fusion proteins biotin ligase acceptor peptide- epidermal growth factor receptor (BLAP-EGFR) and acyl carrier protein - glucosylphosphatidylinositol anchored protein (ACP-CD59)(New England Biolabs, Ipswich, MA) in MEF cells was done by transient co-transfection of four DNA plasmids, BirA-KDEL (1 µg), BLAP-EGFR (1 µg), ACP-CD59 (1 µg), and a plasma membrane fluorescent protein marker, K-Ras2-YFP (0.25 µg, ATCC plasmid 10089283) per 25,000 cells.

Techniques: Diffusion-based Assay, Imaging, Labeling

Journal: PLoS ONE

Article Title: Bridging the Gap between Single Molecule and Ensemble Methods for Measuring Lateral Dynamics in the Plasma Membrane

doi: 10.1371/journal.pone.0078096

Figure Lengend Snippet: Quantitative comparison of kICS and SPT analysis for three QD color set.

Article Snippet: In brief, expression of the fusion proteins biotin ligase acceptor peptide- epidermal growth factor receptor (BLAP-EGFR) and acyl carrier protein - glucosylphosphatidylinositol anchored protein (ACP-CD59)(New England Biolabs, Ipswich, MA) in MEF cells was done by transient co-transfection of four DNA plasmids, BirA-KDEL (1 µg), BLAP-EGFR (1 µg), ACP-CD59 (1 µg), and a plasma membrane fluorescent protein marker, K-Ras2-YFP (0.25 µg, ATCC plasmid 10089283) per 25,000 cells.

Techniques:

Journal: PLoS ONE

Article Title: Bridging the Gap between Single Molecule and Ensemble Methods for Measuring Lateral Dynamics in the Plasma Membrane

doi: 10.1371/journal.pone.0078096

Figure Lengend Snippet: Shown are the results of the time averaged single molecule analysis of the same sub-region as the kICS analysis in . Log 10 (MSD) versus log 10 (τ) plots for all detected single molecule trajectories, N Traj , with length, n>50 image frames, in each separate QD color channel describing the motion of (a) G M1 labeled with ChToxB-QD705s, (b) ACP-CD59 labeled with CoA-QD655s, and (c) BLAP-EGFR labeled with sAv-QD605s. Also shown in each plot is the linear fit to of the average MSD of all trajectories at short time intervals, 1≤n≤5 corresponding to 40≤n τ≤200 ms. (d) Box-and-whisker representation of the fitted diffusion coefficients, D, from all detected single molecule trajectories, N Traj , with length, n>50 image frames and that were statistically best described by the free diffusion model.

Article Snippet: In brief, expression of the fusion proteins biotin ligase acceptor peptide- epidermal growth factor receptor (BLAP-EGFR) and acyl carrier protein - glucosylphosphatidylinositol anchored protein (ACP-CD59)(New England Biolabs, Ipswich, MA) in MEF cells was done by transient co-transfection of four DNA plasmids, BirA-KDEL (1 µg), BLAP-EGFR (1 µg), ACP-CD59 (1 µg), and a plasma membrane fluorescent protein marker, K-Ras2-YFP (0.25 µg, ATCC plasmid 10089283) per 25,000 cells.

Techniques: Labeling, Whisker Assay, Diffusion-based Assay