biotin conjugated cona  (Vector Laboratories)

Journal: bioRxiv

Article Title: Arp2/3 inhibition switches Eps8’s network associations to favour longer actin filament formation necessary for tunneling nanotubes

doi: 10.1101/2022.08.24.504515

Figure Lengend Snippet: ( a ) Experimental setup for pulling membrane nanotubes using a concanavalin A (Con-A)-coated bead (~3 m in diameter) trapped in an optical tweezer (OT). CAD cells stably expressing the small F-actin-binding peptide F-tractin fused to EGFP (green) were exogenously labelled with the lipophilic Cell Mask™ Deep Red plasma membrane stain (magenta). Intensity profiles of the F-actin fluorescence were measured along the nanotube axis and plotted against the nanotube length. ( b, d ) Representative time-lapse images of a nanotube pulled from a DMSO-treated (mock control) (Supplementary Video 5) and a CK-666-treated cell (Supplementary Video 6). White arrowheads annotate the progression of actin development within the nanotube in d . ( c, e ) Plot of actin profiles within pulled nanotubes for mock- and CK-666-treated cells. Insets show a magnified view at the tube extremity to better highlight the greater presence of F-actin in the CK-666 condition as compared to the mock condition. Mock condition, 11 tubes; CK-666 condition, 12 tubes. ( f ) Left: Exponential fits to the actin intensity profiles were performed to determine a characteristic decay length (2 ℓ ) at which the initial intensity at X = 0 decays to a value of 1/ e . For visualization purposes of the analysis, exponential fits are shown for the mean actin profiles computed from the individual plots presented in e and f . Upper and lower limits of the intensity range are shaded. Right: Dot plot of the characteristic decay lengths (2 ℓ ) for mock- and CK-666-treated cells. Data is represented as the mean ± SEM. Mock (11 tubes), 2.78 ± 0.50; CK-666 (12 tubes), 5.80 ± 0.73. Statistical analysis was performed using an unpaired Mann-Whitney test. ( g ) Top: Force plot of a pulled nanotube from a mock-treated cell showing no F-actin development. Solid teal line, 10-point moving average curve. Bottom: Associated images of the indicated time points (g1, g2). ( h ) Top: Force plot of a pulled nanotube from a CK-666-treated cell showing F-actin development spanning the entire nanotube length. Peaks in the force plot (black arrowheads), with magnitudes of Δ F , arise when retrograde flows outcompete actin polymerization (at the nanotube tip) causing bead displacement towards the cell body (recorded as a positive rise in the force in the lab frame). Solid teal line, 10-point moving average curve. Shaded grey region corresponds to a magnified view on the right. Bottom: Associated images of the indicated time points (h1, h2). ( i ) Histogram of the force peak magnitudes (Δ F ). Sample size, 33 peaks. The trapped bead is annotated by a dotted white circle when not clearly visible. Scale bars, 5 μm.

Article Snippet: Beads were then resuspended in PBS to a concentration of 0.05% w/v, and an appropriate amount of a 1 mg mL −1 biotin-conjugated concanavalin A (ConA) solution (C2272, Sigma-Aldrich) was added to the bead suspension assuming a binding capacity of 10 μg ConA per mg of beads.

Techniques: Stable Transfection, Expressing, Binding Assay, Staining, Fluorescence, MANN-WHITNEY

Journal: Frontiers in Chemistry

Article Title: Fluorescence Imaging of Extracellular Potassium Ion Using Potassium Sensing Oligonucleotide

doi: 10.3389/fchem.2022.922094

Figure Lengend Snippet: (A) Extracell imaging by 1 -StAv-ConA, (B) fluorescence ratio of F red/ F green (F red : F.I. of 550–640 nm,/F green : F.I. of 495–540 nm) after adding KCl, λ ex = 488 nm. (C) Extracell imaging by 1 -StAv-sulfo-NHS-biotin, (D) fluorescence ratio of F red/ F green after adding KCl, λ ex = 488 nm. (↑) indicates the change of DMEM medium.

Article Snippet: Two-hundred microliter of 0.5 μM ConA biotin conjugate (Type IV, SIGMA-ALDRICH, 4–8 mol biotin per mol protein) in DMEM (+10% FBS) was added in this dish and incubated for 10 min at 37°C under 5% CO 2 .

Techniques: Imaging, Fluorescence

Journal: Frontiers in Chemistry

Article Title: Fluorescence Imaging of Extracellular Potassium Ion Using Potassium Sensing Oligonucleotide

doi: 10.3389/fchem.2022.922094

Figure Lengend Snippet: (A) Cell surface imaging by 1 -StAv-ConA after adding 10 μM Amphotericin B, 10 μM Bumetanide, and 10 μM Ouabain/DMEM at 11 min (↑), (B) fluorescence ratio of F red/ F green (F red : F.I. of 575–595 nm,/F green : F.I. of 508–528 nm), λ ex = 488 nm in all cells. (C) Extracell imaging by 1 -StAv-sulfo-NHS-biotin and (D) fluorescence ratio of F red/ F green (F red : F.I. of 554–620 nm, F green : F.I. of 500–554 nm), λ ex = 488 nm after adding 26 μM Amphotericin B at 11 min (↑). The three cells shown in (C) were surrounded by ROI (region of interests), and their fluorescence ratios were shown in (D) .

Article Snippet: Two-hundred microliter of 0.5 μM ConA biotin conjugate (Type IV, SIGMA-ALDRICH, 4–8 mol biotin per mol protein) in DMEM (+10% FBS) was added in this dish and incubated for 10 min at 37°C under 5% CO 2 .

Techniques: Imaging, Fluorescence