Journal: European Journal of Biochemistry

Article Title: Analysis of the thyrotropin‐releasing hormone‐degrading ectoenzyme by site‐directed mutagenesis of cysteine residues

doi: 10.1046/j.1432-1327.2000.01277.x

Figure Lengend Snippet: Subcellular distribution of the truncated GFP‐fusion proteins with either cysteine or serine at position 68, as analyzed by confocal laser fluorescence microscopy. BHK cells were transfected either with the empty pEGFP‐N1 vector (A), the EGFP‐tagged, truncated wild‐type cDNA of TRH‐DE (B) or the corresponding C68S mutant (C) and processed as described in .

Article Snippet: We thank André Wolfstein and Beate Sodeik (Institut für physiologische Chemie, Medizinische Hochschule, Hannover) for experienced support with confocal fluorescence microscopy, Lutz Schomburg for advice and helpful discussions, Uwe Grunenberg for excellent technical assistance, Valerie Ashe for linguistic help and the Deutsche Forschungsgemeinschaft for financial support.

Techniques: Fluorescence, Microscopy, Transfection, Plasmid Preparation, Mutagenesis

Journal: JCI Insight

Article Title: A xenogeneic-free system generating functional human gut organoids from pluripotent stem cells

doi: 10.1172/jci.insight.86492

Figure Lengend Snippet: (A) Day 50–60 organoids from human embryonic stem cells (hESCs) (SEES1 cells) immunostained with markers for intestinal differentiation: villin, leucine-rich repeat containing G protein–coupled receptor 5 (LGR5), CDX2, E-cadherin (ECAD), chromogranin A (CGA), mucin-2 (MUC2), defensin α-6, Paneth cell–specific (DEFA6), α-smooth muscle actin (SMA), and protein gene product 9.5 (PGP9.5). Cell nuclei were counterstained with DAPI. PGP9.5-positive enteric neuronal cells within the α-SMA–positive myenteric area are indicated by yellow arrowheads. Scale bar: 50 μm (VILLIN, LGR5, SMA); 100 μm (ECAD and MUC2). (B) An enterocyte with a characteristic brush border (left), and Paneth cells with secretory granules (black arrowhead) and goblet cells containing mucin granules (yellow arrowhead) (right). Scale bar: 10 μm (left); 5 μm (right). (C) Organoids that developed from SEES1 cells expressed EGFP under the LGR5 promoter (green), indicating that they were LGR5-positive gut organoids. Gut tube-like architecture in a day 34 organoid (red square; high-magnification view, white square) (top row). A small number of EGFP-positive cells were detected in the day 34 organoid (white square) by fluorescence microscopy (bottom left). The number of EGFP-positive cells increased at day 41 (bottom right). Scale bar: 300 μm (top left), 100 μm (top right and bottom row).

Article Snippet: Five μM forskolin was added, and organoid morphology was monitored by time-lapse fluorescence laser confocal microscopy (Keyence).

Techniques: Fluorescence, Microscopy

Journal: JCI Insight

Article Title: A xenogeneic-free system generating functional human gut organoids from pluripotent stem cells

doi: 10.1172/jci.insight.86492

Figure Lengend Snippet: (A) Day 115 gut organoids from hESCs (SEES1 cells) immunostained with markers for the cystic fibrosis conductance regulator (CFTR). CFTR was present in the epithelial layers of the gut organoids as well as in the control human intestine. CFTR (green), actin (red), and DAPI (blue). Scale bar: 100 μm. (B) Quantification of forskolin-induced gut organoid swelling. Gut organoids (green) derived from SEES1 cells were monitored by time-lapse fluorescence laser confocal microscopy (Keyence). The organoid surface area (red) was quantified using Hybrid Cell Count BZ-H3C (Keyence). The normalized total organoid surface area was calculated and averaged from 3 individual wells per treatment (n = 3 for each group). A forskolin-induced organoid (19 minutes) was overlaid with a pretreated organoid (0 minutes; 569 μm).

Article Snippet: Five μM forskolin was added, and organoid morphology was monitored by time-lapse fluorescence laser confocal microscopy (Keyence).

Techniques: Control, Derivative Assay, Fluorescence, Confocal Microscopy, Cell Counting

Journal: Drug Delivery

Article Title: AKT2 siRNA delivery with amphiphilic-based polymeric micelles show efficacy against cancer stem cells

doi: 10.1080/10717544.2018.1461276

Figure Lengend Snippet: Internalization profile of PM. (A,B) FACS quantification of micelles uptake by MDA-MB-231 and MCF-7 ALDH1A1 tdTomato positive vs. negative cells. The cells were incubated with the 5-DTAF labeled PM and the percentage of cells emitting green fluorescence (A) as well as the intensity of fluorescence (B) was quantified at different time-points. Results are expressed as mean ± sd ( n = 3), ** p ≤ .01 for de comparison between CSC and non-CSC. (C) Confocal Microscopy analysis of particles internalization containing a fluorescent siRNA (siRNA-AlexaFluor 488). The arrow illustrates the respective intracellular distribution of nanoparticle and Lysotracker DND-99. Note: For better comprehension of the image please visualize the online version of the manuscript.

Article Snippet: In vivo visualization of DiR-PM was performed using a fibered confocal fluorescence microscopy (FCFM) imaging system (Cellvizio ® Dual Band, Mauna Kea Technologies, Paris, France) in patient derived tumor xenografts (PDX).

Techniques: Incubation, Labeling, Fluorescence, Comparison, Confocal Microscopy

Journal: Drug Delivery

Article Title: AKT2 siRNA delivery with amphiphilic-based polymeric micelles show efficacy against cancer stem cells

doi: 10.1080/10717544.2018.1461276

Figure Lengend Snippet: In vivo behavior of PM-siAKT2 in tumors. Real time fibered confocal fluorescence microscopic (FCFM) images of mice administered with DiR labeled PM. Images correspond to representative single frame of live FCFM imaging of tumors of mice administered 30 min prior to imaging (half the dose of DiR-PM) and 4 h before imaging (full dose of DiR-PM). Vasculature was visualized using FITC-Dextran (500 mg/kg). Signal corresponding to DiR-labeled PM is mainly localized into the vessels 30-min post-administration (see arrows), while the DiR signal is more dispersed and distributed 4-h post-administration (asterisk), clearly showing that the PM extravasated from the vessels invading the tumoral parenchyma. Note: For better comprehension of the image please visualize the online version of the manuscript.

Article Snippet: In vivo visualization of DiR-PM was performed using a fibered confocal fluorescence microscopy (FCFM) imaging system (Cellvizio ® Dual Band, Mauna Kea Technologies, Paris, France) in patient derived tumor xenografts (PDX).

Techniques: In Vivo, Fluorescence, Labeling, Imaging