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: bioRxiv

Article Title: Cholesterol recognition motifs in the transmembrane domain of the tyrosine kinase receptor family: the case for TRKB

doi: 10.1101/734012

Figure Lengend Snippet: TRKB and cholesterol interaction. (a,b) Snapshots of the interaction between TRKB and cholesterol at the TMR (see supplement video) indicating that cholesterol interacts with the OH- group (red) in Y433 (blue) in TRKB.TMR (green). (c) The interaction between the TRKB CARC motif and cholesterol happens preferentially at the C-alpha in Y433 residues, as indicated by coarse-grained molecular dynamics simulations. (d) The binding of biotinylated BDNF (bBDNF) and TRKB is modulated by cholesterol in a bell-shaped fashion. Red rectangle area is expanded in (e) for the comparison between BDNF 1ng/ml (black circles) with BDNF 0 (ctrl, open circles). Data expressed as Mean/SEM of (c) cholesterol occupancy; or from binding normalized by (d) chol 0/BDNF10ng/ml or (e) chol 0/BDNF 1ng/ml. ∗p<0.05 from control (BDNF 0 or ctrl, C427).

Article Snippet: The plates were washed 3x with PBS buffer, and a mixture of biotinylated BDNF (bBDNF, 0, 0.1, 1 or 10ng/ml, Alomone Labs, #B-250-B) and cholesterol (0, 20, 50 or 100μM) was added for 1h at room temperature, followed by washing with PBS.

Techniques: Binding Assay