Journal: PLoS ONE

Article Title: Phosphatidylserine Outer Layer Translocation Is Implicated in IL-10 Secretion by Human Regulatory B Cells

doi: 10.1371/journal.pone.0169755

Figure Lengend Snippet: PBMCs were stimulated for 24 hr with CpG/ionomycin/PMA and BFA as described previously and analyzed by FACS for annexin V (AnV) binding and IL-10 and TNFα production in 21 subjects. Cytometry gating is shown in A. Results are presented in percentage of positive cells in B and median of fluorescence (MFI) in C. Wilcoxon’s matched pairs signed rank tests were used. Top of the bar represents the median, and whiskers are IQR 25–75.

Article Snippet: Briefly, 2.10 6 of PBMCs were washed, pre-incubated in annexin binding buffer containing 5 μl of biotinylated Annexin V (BD pharmingen), washed and further incubated with streptavidine.

Techniques: Binding Assay, Cytometry, Fluorescence

Journal: PLoS ONE

Article Title: Phosphatidylserine Outer Layer Translocation Is Implicated in IL-10 Secretion by Human Regulatory B Cells

doi: 10.1371/journal.pone.0169755

Figure Lengend Snippet: A representative plot is presented (A). PBMCs from 12 subjects were analyzed by FACS for B10 precursors phenotype (i.e CD19+CD5+ (B), CD19+CD24 hi CD27 + (C) and CD19+CD24 hi CD38 hi , (D)), and for annexin V binding. Wilcoxon’s matched pairs signed rank tests were used. Data are median (IQR 25–75).

Article Snippet: Briefly, 2.10 6 of PBMCs were washed, pre-incubated in annexin binding buffer containing 5 μl of biotinylated Annexin V (BD pharmingen), washed and further incubated with streptavidine.

Techniques: Binding Assay

Journal: PLoS ONE

Article Title: Phosphatidylserine Outer Layer Translocation Is Implicated in IL-10 Secretion by Human Regulatory B Cells

doi: 10.1371/journal.pone.0169755

Figure Lengend Snippet: PBMCs from 6 healthy subjects were sorted by flow cytometry (FACSARIA) according to annexin V (AnV) staining: AnV positive (AnV + ) and AnV negative B cells (AnV neg ) after exclusion of dead cells (DAPI + )(A). Each B cell subpopulation was then stimulated for B10 generation and analyzed by flow cytometry as previously described (B). (C) shows the % of cytokine-positive cells (IL-10, IL-6, GM-CSF and TNF-a) in AnV + and AnV AnV neg sorted cells. (D) shows IL-10 and IL-6 concentrations assessed by ELISA in supernatant of AnV + and AnV AnV neg sorted cells.

Article Snippet: Briefly, 2.10 6 of PBMCs were washed, pre-incubated in annexin binding buffer containing 5 μl of biotinylated Annexin V (BD pharmingen), washed and further incubated with streptavidine.

Techniques: Flow Cytometry, Staining, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Phosphatidylserine Outer Layer Translocation Is Implicated in IL-10 Secretion by Human Regulatory B Cells

doi: 10.1371/journal.pone.0169755

Figure Lengend Snippet: PBMCs were stained with APC anti-CD19 and FITC conjugated Annexin V, sorted by FACSARIA according to annexin V (AnV) staining: high AnV B cells (AnV hi ); low AnV B cells (AnV low ) and AnV negative B cells (AnV neg ). Cell death was analyzed at 72 hr using DAPI staining for necrosis (A) and DIOC6 (B) for mitochondrial apoptosis (n = 4). Additionally, cell cycle was analyzed using propidium iodide (PI) staining at 72 hr (C), showing the proportion of cells in the subG1 phase (apoptotic cells) and in the S phase (proliferating cells) in AnV+ (AnV hi and AnV low ) and AnV neg B cells (n = 5). Data are median (IQR25-75).

Article Snippet: Briefly, 2.10 6 of PBMCs were washed, pre-incubated in annexin binding buffer containing 5 μl of biotinylated Annexin V (BD pharmingen), washed and further incubated with streptavidine.

Techniques: Staining

Journal: PLoS ONE

Article Title: Phosphatidylserine Outer Layer Translocation Is Implicated in IL-10 Secretion by Human Regulatory B Cells

doi: 10.1371/journal.pone.0169755

Figure Lengend Snippet: PBMCs from 6 healthy subjects were pretreated with biotinylated Annexin V (biot AnV) (n = 8) or with glyburide (Gly) (n = 8) and then stimulated for 24 hr according to the protocol for B10 assessment previously described. A shows representative plots and B shows the results as median (IQR25-75).

Article Snippet: Briefly, 2.10 6 of PBMCs were washed, pre-incubated in annexin binding buffer containing 5 μl of biotinylated Annexin V (BD pharmingen), washed and further incubated with streptavidine.

Techniques:

Journal: ACS nano

Article Title: A Programmable DNA Origami Nanospring That Reports Dynamics of Single Integrin Motion, Force Magnitude and Force Orientation in Living Cells.

doi: 10.1021/acsnano.2c12545

Figure Lengend Snippet: Figure 1. Design of the 4HB DNA NS structure. (a) A cylinder model of the 4HB square lattice (see cross-section). Each cylindrical rod represents a single DNA double helix. Two insertion and deletion sites that produce a superhelical strain are shown in blue and red, respectively. (b) CanDo, a 3D DNA origami structure prediction server,24 predicted a coil structure. Single-stranded DNA (ssDNA) handles projecting out from both ends are shown as red and orange lines, respectively. The complementary ssDNA antihandles are modified with adhesive peptide (cRGDfK, blue hexagon) to the extracellular domain of integrins and biotin (black circle) to attach the NS to the bottom of the dish. (c) An AFM image of the 4HB-NS. The shape was flexible enough to measure single integrin traction force but also showed repetitive bending or expected loops. Scale bar, 200 nm.

Article Snippet: For positive control experiments (cell adhesion with biotinylated cRGDfK), after neutravidin was washed out, 200 μL of biotinylated cRGDfK (1−150 nM in DNA origami buffer, Peptides International, PCI-3697-PI) was added on the glass-bottomed dish, and the mixture was incubated for 10 min. Unbound biotinylated cRGDfK was washed out using a DNA origami buffer.

Techniques: Modification, Adhesive

Journal: ACS nano

Article Title: A Programmable DNA Origami Nanospring That Reports Dynamics of Single Integrin Motion, Force Magnitude and Force Orientation in Living Cells.

doi: 10.1021/acsnano.2c12545

Figure Lengend Snippet: Figure 2. Visualizing NS extensions by single integrins. (a) A typical fluorescence image of Cy3 NS (red) and GFP-paxillin (white) expressed in HFFs and observed by TIRFM. The boxed area is expanded in (b). Scale bar, 10 μm for (a) and 2 μm for (b). The right cartoon in (b) shows NSs attached with integrins via cRGDfK and the glass surface via the biotin−avidin system. (c) A histogram of flattening NS fluorescence spots located outside the cells. The flattening was calculated as 1 −σshort/σlong, where σshort and σlong, respectively, denote the standard deviation for the short and long axes when the fluorescence spot was fit to a 2D Gaussian function. (d, e) Histograms of flattening NS fluorescence spots within (d) and outside FAs underneath cells (e). (n = 370, 428, and 292 molecules from 6, 6, and 5 cells for (c, d, e), respectively.) (f, g) Time trajectories of the angle between the long axis of NS and the horizontal direction of the picture for the spots undergoing Brownian motion (f) and stretching within FAs (g). Each color represents an individual NS.

Article Snippet: For positive control experiments (cell adhesion with biotinylated cRGDfK), after neutravidin was washed out, 200 μL of biotinylated cRGDfK (1−150 nM in DNA origami buffer, Peptides International, PCI-3697-PI) was added on the glass-bottomed dish, and the mixture was incubated for 10 min. Unbound biotinylated cRGDfK was washed out using a DNA origami buffer.

Techniques: Fluorescence, Avidin-Biotin Assay, Standard Deviation

Journal: ACS nano

Article Title: A Programmable DNA Origami Nanospring That Reports Dynamics of Single Integrin Motion, Force Magnitude and Force Orientation in Living Cells.

doi: 10.1021/acsnano.2c12545

Figure Lengend Snippet: Figure 5. NS force sensor indicated dynamics of loaded force and single integrin motions in living cells. (a−c) Representative traces showing the length of the NS (top), loaded force on the NS (middle), and angle of the NS (bottom) for an integrin-bound NS. The force trace was calculated from the NS length. The thin lines indicate raw data; the bold lines indicate moving averages. (d, e) The colors of the traces correspond to (a−c), and the gradations of the colors correspond to the time course. Representative traces showing a single integrin motion tethered with an NS (d) and the force vector (e) in polar coordinates. Colors correspond to (a−c). (f) Histograms of the loaded force distributions for cRGDfK-labeled NS (red, n = 59 molecules; N = 23 cells) and cRGDfK-less NS (blue, n = 138 molecules; N = 24 cells) underneath the cells.

Article Snippet: For positive control experiments (cell adhesion with biotinylated cRGDfK), after neutravidin was washed out, 200 μL of biotinylated cRGDfK (1−150 nM in DNA origami buffer, Peptides International, PCI-3697-PI) was added on the glass-bottomed dish, and the mixture was incubated for 10 min. Unbound biotinylated cRGDfK was washed out using a DNA origami buffer.

Techniques: Plasmid Preparation, Labeling

Journal: Frontiers in Immunology

Article Title: Mouse B cells engineered to express an anti-HPV antibody elicit anti-tumor T cell responses

doi: 10.3389/fimmu.2025.1613879

Figure Lengend Snippet: Immune complexes derived from antibodies secreted by EBCs activate T cells. (A) Isotype-specific ELISA for antibodies against E6 in the supernatants of B cells engineered as in Figure 2A . Mean OD values and SD are shown for IgG (left) or IgM (right) E6-specific antibodies from EBC (orange) or EPOnly (black) cells, n = 2–3. (B) Quantification of IgG data from (A) using a recombinant IgG1 antibody. Mean and SD values are represented. Pooled data from two independent experiments, n = 2. (C) Representative PCR on reverse-transcribed mRNA from EBCs. Primer binding sites are indicated with arrows. The anti-E6 engineering cassette are shown in orange, and endogenous segments in white or gray. For each amplicon, the DNA (top) or mRNA (bottom) are illustrated. Only spliced fragments derived from mRNA are amplified due to polymerization length constraints. (D) Gel electrophoresis of amplicons from (C) , using RNA extracted from EBCS (left) or mRNA from EPOnly control B cells (right). Molecular weight ladders with relative sizes are shown beside each gel. (E) Representative schematic of cell sources used in (F–I) . Bone marrow from naïve mice was used to generate myeloid-derived dendritic cells. T cells were obtained from mice immunized with E6. B cells for engineering were isolated from separate naïve mice. (F) Representative experimental scheme for (G–I) . Myeloid-derived dendritic cells were seeded and loaded with immune complexes formed by incubating E6 antigen with EBC-derived supernatant. T cells from E6-immunized mice were then added to the culture. (G) Intracellular flow cytometry for IFNg in CD8 + T cells from cultures as in (F) , incubated with supernatants from either EBCs (orange) or EPOnly control B cells (black). Pre-gated on singlets, live cells, CD4 − CD8 + . (H) Same as (G) , but for CD4 + CD8 − T cells. (I) ELISA for IFNg in supernatants from cocultures as described in (F) . For (G–I) , mean and SEM are represented, n = 5–7, pooled from three independent experiments. **pv<0.01 for two-way ANOVA with Šidák’s multiple comparisons tests.

Article Snippet: To detect engineering via E6 target epitopes of the respective binders, cells were incubated at 0.5 μM in Cell Staining Buffer (Biolegend) with biotinylated peptides or biotinylated recombinant E6 protein (R&D Systems) after staining for Zombie L/D (Biolegend), washed and stained with streptavidin (Biolegend) and other antibodies for phenotypic characterization.

Techniques: Derivative Assay, Enzyme-linked Immunosorbent Assay, Recombinant, Reverse Transcription, Binding Assay, Amplification, Nucleic Acid Electrophoresis, Control, Molecular Weight, Isolation, Flow Cytometry, Incubation