dio  (Thermo Fisher)


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    Structured Review

    Thermo Fisher dio
    Flow cytometric analysis of spontaneous cell fusion between <t>DiO-labeled</t> tumor cells and DiD-labeled <t>hUCMSCs.</t>
    Dio, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dio/product/Thermo Fisher
    Average 86 stars, based on 40 article reviews
    Price from $9.99 to $1999.99
    dio - by Bioz Stars, 2022-09
    86/100 stars

    Images

    1) Product Images from "Suppression of tumor cell proliferation and migration by human umbilical cord mesenchymal stem cells: A possible role for apoptosis and Wnt signaling"

    Article Title: Suppression of tumor cell proliferation and migration by human umbilical cord mesenchymal stem cells: A possible role for apoptosis and Wnt signaling

    Journal: Oncology Letters

    doi: 10.3892/ol.2018.8368

    Flow cytometric analysis of spontaneous cell fusion between DiO-labeled tumor cells and DiD-labeled hUCMSCs.
    Figure Legend Snippet: Flow cytometric analysis of spontaneous cell fusion between DiO-labeled tumor cells and DiD-labeled hUCMSCs.

    Techniques Used: Labeling

    Confocal laser-scanning images of cell fusion between hUCMSCs and tumor cells. Tumor cells and hUCMSCs were stained with DiO (green) and DiD (red), respectively. All samples were counterstained with Hoechst 33342 (blue) to indicate the nucleus. After co-culture for 72 h, the hUCMSCs were revealed to have merged into A549 or BEL7402 tumor cells, as illustrated by the existence of hybrids with double nuclei and yellow cell membranes (DiO and DiD double-stained cells indicated by the white arrow) in the representative images. Scale bar, 20 µm; magnification, ×400.
    Figure Legend Snippet: Confocal laser-scanning images of cell fusion between hUCMSCs and tumor cells. Tumor cells and hUCMSCs were stained with DiO (green) and DiD (red), respectively. All samples were counterstained with Hoechst 33342 (blue) to indicate the nucleus. After co-culture for 72 h, the hUCMSCs were revealed to have merged into A549 or BEL7402 tumor cells, as illustrated by the existence of hybrids with double nuclei and yellow cell membranes (DiO and DiD double-stained cells indicated by the white arrow) in the representative images. Scale bar, 20 µm; magnification, ×400.

    Techniques Used: Staining, Co-Culture Assay

    2) Product Images from "Fitness of Outer Membrane Vesicles From Komagataeibacter intermedius Is Altered Under the Impact of Simulated Mars-like Stressors Outside the International Space Station"

    Article Title: Fitness of Outer Membrane Vesicles From Komagataeibacter intermedius Is Altered Under the Impact of Simulated Mars-like Stressors Outside the International Space Station

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2020.01268

    A biosafety assessment of EVs after exposure of kombucha multimicrobial culture (KMC) under space/Mars-like stressors outboard the ISS and OMVs of K. intermedius , isolated from post-flight KMCs. (A) A micrograph of OMVs stained with the lipophilic dye DiO visible in murine fibroblasts. Fibroblast nuclei were stained with DAPI. (1, 2) – OMVs of K. intermedius from initial KMC; (3, 4) – OMVs of K. intermedius from top-located KMC (tKMC). Scale bar, 10 μm. (B) The environment level of ʟ-[ 14 C] glutamate in the preparations of rat brain nerve terminals after co-cultivation with EVs from post-flight KMCs. (C) Survival rate of murine embryo fibroblasts after co-cultivation with different concentrations of OMVs from K. intermedius isolated from tKMC. (D) Survival rate of colorectal carcinoma cells COLO 205 after co-cultivation with different concentrations of OMVs/ K. intermedius , isolated from KMCs exposed outboard the ISS. (E) Endotoxin activity of EVs/KMCs detected with the Limulus amebocyte lysate (LAL) assay compared to standard endotoxin activity from Escherichia coli . (F) Endotoxin activity of OMVs/ K. intermedius detected with the LAL assay. Data were shown as mean ± SD ( n = 3), * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.
    Figure Legend Snippet: A biosafety assessment of EVs after exposure of kombucha multimicrobial culture (KMC) under space/Mars-like stressors outboard the ISS and OMVs of K. intermedius , isolated from post-flight KMCs. (A) A micrograph of OMVs stained with the lipophilic dye DiO visible in murine fibroblasts. Fibroblast nuclei were stained with DAPI. (1, 2) – OMVs of K. intermedius from initial KMC; (3, 4) – OMVs of K. intermedius from top-located KMC (tKMC). Scale bar, 10 μm. (B) The environment level of ʟ-[ 14 C] glutamate in the preparations of rat brain nerve terminals after co-cultivation with EVs from post-flight KMCs. (C) Survival rate of murine embryo fibroblasts after co-cultivation with different concentrations of OMVs from K. intermedius isolated from tKMC. (D) Survival rate of colorectal carcinoma cells COLO 205 after co-cultivation with different concentrations of OMVs/ K. intermedius , isolated from KMCs exposed outboard the ISS. (E) Endotoxin activity of EVs/KMCs detected with the Limulus amebocyte lysate (LAL) assay compared to standard endotoxin activity from Escherichia coli . (F) Endotoxin activity of OMVs/ K. intermedius detected with the LAL assay. Data were shown as mean ± SD ( n = 3), * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.

    Techniques Used: Isolation, Staining, Activity Assay, LAL Assay

    3) Product Images from "The Dual Effect of Rho-Kinase Inhibition on Trabecular Meshwork Cells Cytoskeleton and Extracellular Matrix in an In Vitro Model of Glaucoma"

    Article Title: The Dual Effect of Rho-Kinase Inhibition on Trabecular Meshwork Cells Cytoskeleton and Extracellular Matrix in an In Vitro Model of Glaucoma

    Journal: Journal of Clinical Medicine

    doi: 10.3390/jcm11041001

    Confocal microscopy images of the 3D cultured pHTMC. Analysis of the effect of the ROCK inhibitor Y27632 at 25 nM for 24 h and the effect of LT at 0.5 μg/mL on primary human pHTMC treated with TGF-β2 at 5 ng/mL for 48 h. The cells were treated with TMCM alone (vehicle), 5 ng/mL of TGF-β2 for 48 h, or with TGF-β2 (5 ng/mL) for 24 h followed by a combination of TGF-β2 at 5 ng/mL and with Y-27632 (25 nM) or LT (0.5 µg/mL) for 24 h. Actin fibers are stained in red by phalloidin, membranes with DiO (green), and nuclei with DAPI (blue). Magnification 200×. Scale bar = 30 µm.
    Figure Legend Snippet: Confocal microscopy images of the 3D cultured pHTMC. Analysis of the effect of the ROCK inhibitor Y27632 at 25 nM for 24 h and the effect of LT at 0.5 μg/mL on primary human pHTMC treated with TGF-β2 at 5 ng/mL for 48 h. The cells were treated with TMCM alone (vehicle), 5 ng/mL of TGF-β2 for 48 h, or with TGF-β2 (5 ng/mL) for 24 h followed by a combination of TGF-β2 at 5 ng/mL and with Y-27632 (25 nM) or LT (0.5 µg/mL) for 24 h. Actin fibers are stained in red by phalloidin, membranes with DiO (green), and nuclei with DAPI (blue). Magnification 200×. Scale bar = 30 µm.

    Techniques Used: Confocal Microscopy, Cell Culture, Staining

    4) Product Images from "Cleavage of galectin-3 by matrix metalloproteases induces angiogenesis in breast cancer"

    Article Title: Cleavage of galectin-3 by matrix metalloproteases induces angiogenesis in breast cancer

    Journal: International Journal of Cancer. Journal International du Cancer

    doi: 10.1002/ijc.25254

    Cell migration using wound healing assay. ( a ) BAMEC and BT-549-H/P 64 cells were prelabeled with DiO and DiI, respectively, and seeded in each chamber of cell culture insert. After 24 hr, the inserts were removed and cell migration was studied. ( a′–a′″
    Figure Legend Snippet: Cell migration using wound healing assay. ( a ) BAMEC and BT-549-H/P 64 cells were prelabeled with DiO and DiI, respectively, and seeded in each chamber of cell culture insert. After 24 hr, the inserts were removed and cell migration was studied. ( a′–a′″

    Techniques Used: Migration, Wound Healing Assay, Cell Culture

    5) Product Images from "Selective apoptosis of pluripotent mouse and human stem cells by novel ceramide analogues prevents teratoma formation and enriches for neural precursors in ES cell-derived neural transplants"

    Article Title: Selective apoptosis of pluripotent mouse and human stem cells by novel ceramide analogues prevents teratoma formation and enriches for neural precursors in ES cell-derived neural transplants

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200405144

    EBCs from untreated EBs form highly invasive cortical and ventricular tumors, whereas S18-treated EBCs show enhanced neuronal differentiation after engraftment. (A) A tumor developed from Vybrant CM diI-labeled ROSA-26 EBCs was immunostained for β-galactosidase (Cy2, green). The arrow indicates a residual cluster of Vybrant CM diI-labeled cells. (B) S18-treated or untreated EBCs were stained with Vybrant CM diI (red, untreated cells) or Vybrant diO (green, treated cells), mixed, and injected into the striatum of neonatal mice. The figure shows settlement of treated cells in the subependymal layer, whereas untreated EBCs form a neural tube-like tumor in the lumen of the right lateral ventricle. (C) Mouse EBCs derived from S18-treated EBs were injected into the striatum of neonatal mice and immunostained for nestin (Cy3, red). (D) After nestin staining, frozen sections were FISH-stained for Y-chromosomes (FITC, green). DNA was counterstained with Hoechst dye (blue). (E) Mouse EBs were treated with 80 μM of S18, labeled with Vybrant CM diI (red), and injected into the striatum of neonatal mice. 6 wk after engraftment, brain sections were immunostained for β-tubulin III (Cy2, green). Arrrow shows cluster of Vybrant CM diI-positive (red) cells that are double-stained for β-tubulin III (Cy2, cryosectioned, confocal).
    Figure Legend Snippet: EBCs from untreated EBs form highly invasive cortical and ventricular tumors, whereas S18-treated EBCs show enhanced neuronal differentiation after engraftment. (A) A tumor developed from Vybrant CM diI-labeled ROSA-26 EBCs was immunostained for β-galactosidase (Cy2, green). The arrow indicates a residual cluster of Vybrant CM diI-labeled cells. (B) S18-treated or untreated EBCs were stained with Vybrant CM diI (red, untreated cells) or Vybrant diO (green, treated cells), mixed, and injected into the striatum of neonatal mice. The figure shows settlement of treated cells in the subependymal layer, whereas untreated EBCs form a neural tube-like tumor in the lumen of the right lateral ventricle. (C) Mouse EBCs derived from S18-treated EBs were injected into the striatum of neonatal mice and immunostained for nestin (Cy3, red). (D) After nestin staining, frozen sections were FISH-stained for Y-chromosomes (FITC, green). DNA was counterstained with Hoechst dye (blue). (E) Mouse EBs were treated with 80 μM of S18, labeled with Vybrant CM diI (red), and injected into the striatum of neonatal mice. 6 wk after engraftment, brain sections were immunostained for β-tubulin III (Cy2, green). Arrrow shows cluster of Vybrant CM diI-positive (red) cells that are double-stained for β-tubulin III (Cy2, cryosectioned, confocal).

    Techniques Used: Labeling, Staining, Injection, Mouse Assay, Derivative Assay, Fluorescence In Situ Hybridization

    6) Product Images from "Ventrolateral Origin of Each Cycle of Rhythmic Activity Generated by the Spinal Cord of the Chick Embryo"

    Article Title: Ventrolateral Origin of Each Cycle of Rhythmic Activity Generated by the Spinal Cord of the Chick Embryo

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0000417

    Antidromic stimulation of the ventral roots to identify the location of the motor nucleus. A. The optical responses recorded from the photodiode array are shown superimposed over a confocal transverse section of the slice in which the recordings were made. After the optical experiment, the slice was fixed and motoneurons were labeled retrogradely with DiI (green) applied to the ventral root and primary afferents were labeled anterogradely with DiO (red) applied to the dorsal root. Each optical signal is the response to a single antidromic stimulus averaged from a train of stimuli (8 stimuli at 20 Hz) and recorded in the presence of a cholinergic (mecamylamine 50 µM) and a GABAA (bicuculline 50 µM) antagonist to block the synaptic inputs from R-interneurons onto motoneurons (33). The largest antidromic responses coincided with the location of motoneuron cell bodies. B. Antidromically-evoked optical responses from another cord in which it was possible to detect antidromic spikes adjacent to the central canal at a location corresponding to that of preganglionic neurons (red traces), in addition to those over the motor nucleus (green traces). DR-dorsal root; VR-Ventral Root. The colored traces in panel B have been averaged and are displayed on an expanded scale in C. It can be seen that the antidromic responses near the central canal are smaller and have a longer latency that those recorded from the motor nucleus. D. The slow antidromic responses recorded over motoneurons are depressed in the presence of the nicotinic cholinergic antagonist mecamylamine and the GABAA receptor antagonist bicuculline to block the recurrent synaptic input from R-interneurons to motoneurons. The upper trace (control) was obtained under control conditions and the lower trace (bicuculline and mecamylamine) in the presence of the drugs. The time of the ventral root stimuli (stim.) are marked by the dots beneath the traces. E. Spectrum of optical responses recorded over motoneurons and evoked by ventral root stimulation under control conditions. The signals were obtained at three different illumination wavelengths in response to a train of ventral root stimuli (stim.). Note the reversal of the signals at 580 nm and the absence of a significant signal at 630 nm, the isosbestic point of the dye. The times of the ventral root stimuli (stim.) are indicated beneath the traces. In this, and all other figures, the vertical calibration arrows indicate the direction of increased light transmission (decreased light absorption). Data in A from an E11 embryo and in B, C and D from an E10 embryo. Data in E from an E11 embryo.
    Figure Legend Snippet: Antidromic stimulation of the ventral roots to identify the location of the motor nucleus. A. The optical responses recorded from the photodiode array are shown superimposed over a confocal transverse section of the slice in which the recordings were made. After the optical experiment, the slice was fixed and motoneurons were labeled retrogradely with DiI (green) applied to the ventral root and primary afferents were labeled anterogradely with DiO (red) applied to the dorsal root. Each optical signal is the response to a single antidromic stimulus averaged from a train of stimuli (8 stimuli at 20 Hz) and recorded in the presence of a cholinergic (mecamylamine 50 µM) and a GABAA (bicuculline 50 µM) antagonist to block the synaptic inputs from R-interneurons onto motoneurons (33). The largest antidromic responses coincided with the location of motoneuron cell bodies. B. Antidromically-evoked optical responses from another cord in which it was possible to detect antidromic spikes adjacent to the central canal at a location corresponding to that of preganglionic neurons (red traces), in addition to those over the motor nucleus (green traces). DR-dorsal root; VR-Ventral Root. The colored traces in panel B have been averaged and are displayed on an expanded scale in C. It can be seen that the antidromic responses near the central canal are smaller and have a longer latency that those recorded from the motor nucleus. D. The slow antidromic responses recorded over motoneurons are depressed in the presence of the nicotinic cholinergic antagonist mecamylamine and the GABAA receptor antagonist bicuculline to block the recurrent synaptic input from R-interneurons to motoneurons. The upper trace (control) was obtained under control conditions and the lower trace (bicuculline and mecamylamine) in the presence of the drugs. The time of the ventral root stimuli (stim.) are marked by the dots beneath the traces. E. Spectrum of optical responses recorded over motoneurons and evoked by ventral root stimulation under control conditions. The signals were obtained at three different illumination wavelengths in response to a train of ventral root stimuli (stim.). Note the reversal of the signals at 580 nm and the absence of a significant signal at 630 nm, the isosbestic point of the dye. The times of the ventral root stimuli (stim.) are indicated beneath the traces. In this, and all other figures, the vertical calibration arrows indicate the direction of increased light transmission (decreased light absorption). Data in A from an E11 embryo and in B, C and D from an E10 embryo. Data in E from an E11 embryo.

    Techniques Used: Labeling, Blocking Assay, Transmission Assay

    Timing of voltage-sensitive dye signals recorded from different regions of the cord at the onset of individual cycles. A. Electrical and optical recordings from an E10 embryo during an episode initiated by a dorsal root stimulus. The optical recording is from a single diode over the later motor column ipsilateral to the neural recordings. The dotted grey lines demarcate the onset of discharge in each cycle of activity. B. Electrical (black) and optical recordings from 3 different regions of the cord. As in previous figures, the red traces were averaged from diodes over the lateral motor column, the green traces from over the intermediate region and the blue traces from over the dorsal region. The diodes and their locations are shown in panel D. The neurogram is the DC trace low pass filtered between DC-20Hz. The episode was triggered by a single stimulus to the ipsilateral dorsal root at the time marked by the arrow (S). The numbered cycles correspond to those in panel A. The initial part of the first cycle (demarcated by a gray box) has been blown up in the inset to show the timing of the earliest optical and electrical activity. C. Quantification of the timing of optical activity in each cycle with respect to the onset of the electrical activity recorded from the ventral root. The onset delay of the optical signals with respect to the onset of the electrical signal is plotted for each cycle for each of the three regions. The numbers beside the plots correspond to the numbered cycles shown in A and B. Note that the dorsal optical activity precedes that of the electrical activity in the first cycle. Moreover the first cycle exhibits a dorsoventral sequence of activation but all of the other cycles are activated ventrodorsally. D. Location of the diodes whose signals were averaged to produce the traces shown in panels B and E. The diode signals are superimposed over a stained section in which the motoneurons were labeled with DiO and the dorsal root afferents were labeled with DiI. E. Cycle-triggered, averaged DC ventral root potential (VR DC-smoothed with a 20 point moving average) and integrated ventral root discharge (VR INT) together with the optical responses from the three different cord regions ipsilateral to the earliest ventral root activity. The last three cycles (3–5 in A) were averaged for these records. As before, each trace was averaged from several adjacent diodes (shown in D) and was normalized to its peak amplitude. Data were obtained at a sampling interval of 1.011 ms. F. Montage showing the pseudocolored diode array signals superimposed on the outline of the cord slice. Each image is the average of 45 frames obtained at the times indicated by the numbered regions over the traces in E. The first panel in the sequence (Anti) is an image generated during antidromic stimulation of the ventral root to identify the location of the lateral motor column. The arrow in image 3 shows the initial activity within the lateral motor column. The last image in the series (pre) was averaged from the frames delimited by the red rectangle marked pre in panel A. This image shows the spatial distribution of activity just before the last cycle.
    Figure Legend Snippet: Timing of voltage-sensitive dye signals recorded from different regions of the cord at the onset of individual cycles. A. Electrical and optical recordings from an E10 embryo during an episode initiated by a dorsal root stimulus. The optical recording is from a single diode over the later motor column ipsilateral to the neural recordings. The dotted grey lines demarcate the onset of discharge in each cycle of activity. B. Electrical (black) and optical recordings from 3 different regions of the cord. As in previous figures, the red traces were averaged from diodes over the lateral motor column, the green traces from over the intermediate region and the blue traces from over the dorsal region. The diodes and their locations are shown in panel D. The neurogram is the DC trace low pass filtered between DC-20Hz. The episode was triggered by a single stimulus to the ipsilateral dorsal root at the time marked by the arrow (S). The numbered cycles correspond to those in panel A. The initial part of the first cycle (demarcated by a gray box) has been blown up in the inset to show the timing of the earliest optical and electrical activity. C. Quantification of the timing of optical activity in each cycle with respect to the onset of the electrical activity recorded from the ventral root. The onset delay of the optical signals with respect to the onset of the electrical signal is plotted for each cycle for each of the three regions. The numbers beside the plots correspond to the numbered cycles shown in A and B. Note that the dorsal optical activity precedes that of the electrical activity in the first cycle. Moreover the first cycle exhibits a dorsoventral sequence of activation but all of the other cycles are activated ventrodorsally. D. Location of the diodes whose signals were averaged to produce the traces shown in panels B and E. The diode signals are superimposed over a stained section in which the motoneurons were labeled with DiO and the dorsal root afferents were labeled with DiI. E. Cycle-triggered, averaged DC ventral root potential (VR DC-smoothed with a 20 point moving average) and integrated ventral root discharge (VR INT) together with the optical responses from the three different cord regions ipsilateral to the earliest ventral root activity. The last three cycles (3–5 in A) were averaged for these records. As before, each trace was averaged from several adjacent diodes (shown in D) and was normalized to its peak amplitude. Data were obtained at a sampling interval of 1.011 ms. F. Montage showing the pseudocolored diode array signals superimposed on the outline of the cord slice. Each image is the average of 45 frames obtained at the times indicated by the numbered regions over the traces in E. The first panel in the sequence (Anti) is an image generated during antidromic stimulation of the ventral root to identify the location of the lateral motor column. The arrow in image 3 shows the initial activity within the lateral motor column. The last image in the series (pre) was averaged from the frames delimited by the red rectangle marked pre in panel A. This image shows the spatial distribution of activity just before the last cycle.

    Techniques Used: Activity Assay, Sequencing, Activation Assay, Staining, Labeling, Sampling, Mass Spectrometry, Generated

    7) Product Images from "Interpreting the pathogenicity of Joubert syndrome missense variants in Caenorhabditis elegans"

    Article Title: Interpreting the pathogenicity of Joubert syndrome missense variants in Caenorhabditis elegans

    Journal: Disease Models & Mechanisms

    doi: 10.1242/dmm.046631

    P74S and G155S mutations in mksr-2 differentially disrupt cilium structure and function. (A) Representative images of DiI staining in head and tail ciliated sensory neurons of various mksr-2 alleles in either nphp-4(+) or nphp-4(Δ) genetic backgrounds. Dashed line indicates the worm body. Anterior is to the left. (B) Histogram showing the frequency of worms with DiO uptake in zero to four phasmid neurons. Data were combined from three independent experiments. n , number of worms. (C) Assessment of phasmid cilium length using an XBX-1::tdTomato reporter. White asterisks denote short cilia. Black lines are the mean±s.d. Total number of cilia: WT ( n =41), mksr-2 ( n =55), mNG::mksr-2(+) ( n =48), mNG::mksr-2(P74S) ( n =41), mNG::mksr-2(G155S) ( n =50), nphp-4 ( n =39), nphp-4;mksr-2 ( n =63), nphp-4;mNG::mksr-2(+) ( n =44), nphp-4;mNG::mksr-2(P74S) ( n =47), nphp-4;mNG::mksr-2(G155S) ( n =53). Statistical significance was assessed by one-way ANOVA followed by Tukey's post hoc test [ P -values: mNG::mksr-2(+) versus mNG::mksr-2(G155S) , P =0.032; nphp-4 versus nphp-4;mksr-2 , P =9.1×10 −15 ; nphp-4;mNG::mksr-2(+) versus nphp-4;mNG::mksr-2(P74S) , P =8.9×10 −15 ; nphp-4;mNG::mksr-2(+) versus nphp-4;mNG::mksr-2(G155S) , P =9.1×10 −15 ]. (D) Assessment of dendrite length using an XBX-1::tdTomato reporter. Dashed lines denote the dendrites. Anterior is to the left. Black lines are the mean±s.d. Total number of dendrites: WT ( n =31), mksr-2 ( n =53), mNG::mksr-2(+) ( n =39), mNG::mksr-2(P74S) ( n =32), mNG::mksr-2(G155S) ( n =43), nphp-4 ( n =34), nphp-4;mksr-2 ( n =53), nphp-4;mNG::mksr-2(+) ( n =45), nphp-4;mNG::mksr-2(P74S) ( n =44), nphp-4;mNG::mksr-2(G155S) ( n =58). Statistical significance was assessed by one-way ANOVA followed by Tukey's post hoc test [ P -values: mNG::mksr-2(+) versus mNG::mksr-2(G155S) , P =4.7×10 −6 ; nphp-4 versus nphp-4;mksr-2 , P =2.4×10 −14 ; nphp-4;mNG::mksr-2(+) versus nphp-4;mNG::mksr-2(P74S) , P =2.6×10 −8 ; nphp-4;mNG::mksr-2(+) versus nphp-4;mNG::mksr-2(G155S) , P =2.3×10 −14 ; nphp-4;mNG::mksr-2(P74S) versus nphp-4;mNG::mksr-2(G155S) , P =0.012]. (E) Assessment of amphid cilia clustering using the XBX-1::tdTomato reporter. White asterisks indicate amphid neuron basal bodies (and cilia) that are mispositioned, proximal to the main cluster. Anterior is to the right. Black lines are the mean±s.d. Total number of worms: WT ( n =37), mksr-2 ( n =34), mNG::mksr-2(+) ( n =41), mNG::mksr-2(P74S) ( n =43), mNG::mksr-2(G155S) ( n =40), nphp-4 ( n =40), nphp-4;mksr-2 ( n =44), nphp-4;mNG::mksr-2(+) ( n =26), nphp-4;mNG::mksr-2(P74S) ( n =44), nphp-4;mNG::mksr-2(G155S) ( n =37). Statistical significance was assessed by Kruskal–Wallis test followed by Dunn's post hoc test [ P -values: WT versus mksr-2 , P =4.9×10 −8 ; mNG::mksr-2(+) versus mNG::mksr-2(G155S) , P =0.0012; nphp-4 versus nphp-4;mksr-2 , P =6.7×10 −16 ; nphp-4;mNG::mksr-2(+) versus nphp-4;mNG::mksr-2(P74S) , P =1.2×10 −8 ; nphp-4;mNG::mksr-2(+) versus nphp-4;mNG::mksr-2(G155S) , P =1.7×10 −7 ]. (F) Assessment of worm roaming behaviour normalised to WT. Data were pooled from three independent experiments. Black lines are the mean±s.d. Total number of worms: WT ( n =46), mksr-2 ( n =44), mNG::mksr- 2(+) ( n =44), mNG::mksr-2(P74S) ( n =43), mNG::mksr-2(G155S) ( n =44), nphp-4 ( n =44), nphp-4;mksr-2 ( n =43), nphp-4;mNG::mksr-2(+) ( n =44), nphp-4;mNG::mksr-2(P74S) ( n =43), nphp-4;mNG::mksr-2(G155S) ( n =46). Statistical significance was assessed by one-way ANOVA followed by Tukey's post hoc test [ P -values: WT versus mksr-2 , P =5.4×10 −10 ; nphp-4 versus nphp-4;mksr-2 , P =1.8×10 −11 ; nphp-4;mNG::mksr-2(+) versus nphp-4;mNG::mksr-2(P74S) , P =3.1×10 −14 ; nphp-4;mNG::mksr-2(+) versus nphp-4;mNG::mksr-2(G155S) , P =3.1×10 −14 ]. (G) Assessment of osmotic avoidance behaviour in a 10-min assay. For each genotype, n =18. Statistical significance was assessed by Kruskal–Wallis test followed by Dunn's post hoc test at 10 min [ P -values: nphp-4 versus nphp-4;mksr-2 , P =9.8×10 −11 ; nphp-4;mNG::mksr-2(+) versus nphp-4;mNG::mksr-2(P74S) , P =0.0007; nphp-4;mNG::mksr-2(+) versus nphp-4;mNG::mksr-2(G155S) , P =3.8×10 −7 ]. * P
    Figure Legend Snippet: P74S and G155S mutations in mksr-2 differentially disrupt cilium structure and function. (A) Representative images of DiI staining in head and tail ciliated sensory neurons of various mksr-2 alleles in either nphp-4(+) or nphp-4(Δ) genetic backgrounds. Dashed line indicates the worm body. Anterior is to the left. (B) Histogram showing the frequency of worms with DiO uptake in zero to four phasmid neurons. Data were combined from three independent experiments. n , number of worms. (C) Assessment of phasmid cilium length using an XBX-1::tdTomato reporter. White asterisks denote short cilia. Black lines are the mean±s.d. Total number of cilia: WT ( n =41), mksr-2 ( n =55), mNG::mksr-2(+) ( n =48), mNG::mksr-2(P74S) ( n =41), mNG::mksr-2(G155S) ( n =50), nphp-4 ( n =39), nphp-4;mksr-2 ( n =63), nphp-4;mNG::mksr-2(+) ( n =44), nphp-4;mNG::mksr-2(P74S) ( n =47), nphp-4;mNG::mksr-2(G155S) ( n =53). Statistical significance was assessed by one-way ANOVA followed by Tukey's post hoc test [ P -values: mNG::mksr-2(+) versus mNG::mksr-2(G155S) , P =0.032; nphp-4 versus nphp-4;mksr-2 , P =9.1×10 −15 ; nphp-4;mNG::mksr-2(+) versus nphp-4;mNG::mksr-2(P74S) , P =8.9×10 −15 ; nphp-4;mNG::mksr-2(+) versus nphp-4;mNG::mksr-2(G155S) , P =9.1×10 −15 ]. (D) Assessment of dendrite length using an XBX-1::tdTomato reporter. Dashed lines denote the dendrites. Anterior is to the left. Black lines are the mean±s.d. Total number of dendrites: WT ( n =31), mksr-2 ( n =53), mNG::mksr-2(+) ( n =39), mNG::mksr-2(P74S) ( n =32), mNG::mksr-2(G155S) ( n =43), nphp-4 ( n =34), nphp-4;mksr-2 ( n =53), nphp-4;mNG::mksr-2(+) ( n =45), nphp-4;mNG::mksr-2(P74S) ( n =44), nphp-4;mNG::mksr-2(G155S) ( n =58). Statistical significance was assessed by one-way ANOVA followed by Tukey's post hoc test [ P -values: mNG::mksr-2(+) versus mNG::mksr-2(G155S) , P =4.7×10 −6 ; nphp-4 versus nphp-4;mksr-2 , P =2.4×10 −14 ; nphp-4;mNG::mksr-2(+) versus nphp-4;mNG::mksr-2(P74S) , P =2.6×10 −8 ; nphp-4;mNG::mksr-2(+) versus nphp-4;mNG::mksr-2(G155S) , P =2.3×10 −14 ; nphp-4;mNG::mksr-2(P74S) versus nphp-4;mNG::mksr-2(G155S) , P =0.012]. (E) Assessment of amphid cilia clustering using the XBX-1::tdTomato reporter. White asterisks indicate amphid neuron basal bodies (and cilia) that are mispositioned, proximal to the main cluster. Anterior is to the right. Black lines are the mean±s.d. Total number of worms: WT ( n =37), mksr-2 ( n =34), mNG::mksr-2(+) ( n =41), mNG::mksr-2(P74S) ( n =43), mNG::mksr-2(G155S) ( n =40), nphp-4 ( n =40), nphp-4;mksr-2 ( n =44), nphp-4;mNG::mksr-2(+) ( n =26), nphp-4;mNG::mksr-2(P74S) ( n =44), nphp-4;mNG::mksr-2(G155S) ( n =37). Statistical significance was assessed by Kruskal–Wallis test followed by Dunn's post hoc test [ P -values: WT versus mksr-2 , P =4.9×10 −8 ; mNG::mksr-2(+) versus mNG::mksr-2(G155S) , P =0.0012; nphp-4 versus nphp-4;mksr-2 , P =6.7×10 −16 ; nphp-4;mNG::mksr-2(+) versus nphp-4;mNG::mksr-2(P74S) , P =1.2×10 −8 ; nphp-4;mNG::mksr-2(+) versus nphp-4;mNG::mksr-2(G155S) , P =1.7×10 −7 ]. (F) Assessment of worm roaming behaviour normalised to WT. Data were pooled from three independent experiments. Black lines are the mean±s.d. Total number of worms: WT ( n =46), mksr-2 ( n =44), mNG::mksr- 2(+) ( n =44), mNG::mksr-2(P74S) ( n =43), mNG::mksr-2(G155S) ( n =44), nphp-4 ( n =44), nphp-4;mksr-2 ( n =43), nphp-4;mNG::mksr-2(+) ( n =44), nphp-4;mNG::mksr-2(P74S) ( n =43), nphp-4;mNG::mksr-2(G155S) ( n =46). Statistical significance was assessed by one-way ANOVA followed by Tukey's post hoc test [ P -values: WT versus mksr-2 , P =5.4×10 −10 ; nphp-4 versus nphp-4;mksr-2 , P =1.8×10 −11 ; nphp-4;mNG::mksr-2(+) versus nphp-4;mNG::mksr-2(P74S) , P =3.1×10 −14 ; nphp-4;mNG::mksr-2(+) versus nphp-4;mNG::mksr-2(G155S) , P =3.1×10 −14 ]. (G) Assessment of osmotic avoidance behaviour in a 10-min assay. For each genotype, n =18. Statistical significance was assessed by Kruskal–Wallis test followed by Dunn's post hoc test at 10 min [ P -values: nphp-4 versus nphp-4;mksr-2 , P =9.8×10 −11 ; nphp-4;mNG::mksr-2(+) versus nphp-4;mNG::mksr-2(P74S) , P =0.0007; nphp-4;mNG::mksr-2(+) versus nphp-4;mNG::mksr-2(G155S) , P =3.8×10 −7 ]. * P

    Techniques Used: Staining

    8) Product Images from "Central and Peripheral Retina Arise through Distinct Developmental Paths"

    Article Title: Central and Peripheral Retina Arise through Distinct Developmental Paths

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0061422

    The Peripheral not Central Optic Cup Originates in the Distal Optic Vesicle. A: Scheme of the predicted distribution of dye directed to the OV of an HH10 embryo. The distal-most tip (red) is predicted to generate central neural retina. Central RPE is predicted to arise from the dorsal OV (yellow dots). OCL (green dots) origin is not known B: Dorsal view of a 12-somite embryo after DiI targeted to the distal OV (red arrow). C: Lateral view of the embryo in B following re-incubation. Dye distributed to the dorsal eye, from the OCL towards the central eye (white bar). Asterisk marks dye in the ectoderm. Pink line marks boundary between lens and OCL. D–G: Coronal sections of the embryo in B, C. D: Lower magnification for orientation. E–F: Dye in the dorsal OCL. Dye is present in the OCL and adjacent inner and outer layers (arrows). G: Dye is absent in both central neural retinal and RPE. H: DiI (red) and DiO (green) targeted to anterior and posterior distal OV. I: The embryo in H following re-incubation. DiO is distributed around the temporal OCL (green arrows) and DiI around the nasal OCL (red arrows). J–K: Transverse section through an embryo targeted at the anterior distal OV. Dye is restricted to the OCL and into the peripheral inner and outer layers (arrow). L: Graph showing dye distribution in the optic cup following distal OV labeling. Most embryos showed dye distributed to the OCL and excluded from the central neural retina or RPE. M: Scheme of the measuring strategy to analyze dye distribution. N: Plot of the distribution of distal OV targets against the midpoint of dye distribution around the OCL. Targeting through the posterior to anterior of the distal OV trends from temporal to nasal OCL. NR-neural retina, RPE-retinal pigmented epithelium, OCL-optic cup lip, L-lens, A-anterior, P-posterior, Pr-proximal, Di-distal, D-dorsal, V-ventral, T-temporal, N-nasal, OV-optic vesicle, OCL-optic cup lip. Scale bars = 100 µm.
    Figure Legend Snippet: The Peripheral not Central Optic Cup Originates in the Distal Optic Vesicle. A: Scheme of the predicted distribution of dye directed to the OV of an HH10 embryo. The distal-most tip (red) is predicted to generate central neural retina. Central RPE is predicted to arise from the dorsal OV (yellow dots). OCL (green dots) origin is not known B: Dorsal view of a 12-somite embryo after DiI targeted to the distal OV (red arrow). C: Lateral view of the embryo in B following re-incubation. Dye distributed to the dorsal eye, from the OCL towards the central eye (white bar). Asterisk marks dye in the ectoderm. Pink line marks boundary between lens and OCL. D–G: Coronal sections of the embryo in B, C. D: Lower magnification for orientation. E–F: Dye in the dorsal OCL. Dye is present in the OCL and adjacent inner and outer layers (arrows). G: Dye is absent in both central neural retinal and RPE. H: DiI (red) and DiO (green) targeted to anterior and posterior distal OV. I: The embryo in H following re-incubation. DiO is distributed around the temporal OCL (green arrows) and DiI around the nasal OCL (red arrows). J–K: Transverse section through an embryo targeted at the anterior distal OV. Dye is restricted to the OCL and into the peripheral inner and outer layers (arrow). L: Graph showing dye distribution in the optic cup following distal OV labeling. Most embryos showed dye distributed to the OCL and excluded from the central neural retina or RPE. M: Scheme of the measuring strategy to analyze dye distribution. N: Plot of the distribution of distal OV targets against the midpoint of dye distribution around the OCL. Targeting through the posterior to anterior of the distal OV trends from temporal to nasal OCL. NR-neural retina, RPE-retinal pigmented epithelium, OCL-optic cup lip, L-lens, A-anterior, P-posterior, Pr-proximal, Di-distal, D-dorsal, V-ventral, T-temporal, N-nasal, OV-optic vesicle, OCL-optic cup lip. Scale bars = 100 µm.

    Techniques Used: Incubation, Labeling

    Identification of Central Optic Cup Fields in the Optic Vesicle. A: Schematic dorsal view of an optic vesicle at HH9/10 summarizing non-distal OV zones. B: Dye targeted to the posterior OV (arrow). C–E: Coronal sections of B following re-incubation. C: Low magnification. D–E: Higher magnification showing dye distributed in the central neural retina (red arrows). Dye is excluded from the OCL and posterior RPE. F: Dorsal view with DiI at the distal OV (red arrow) and DiO proximal (green arrows). G–I: Coronal section through the embryo in F following reincubation. G: DiI is distributed to the OCL. The central limit of DiI distribution is indicated (red arrow). H: DiO is distributed through the central RPE. The peripheral limit of DiO distribution is indicated (green arrow). I: Composite of G/H. DiI (red) in the OCL and DiO (green) in the central RPE do not overlap. J: Dorsal view of dye label proximal in the OV (red arrow). K: The same embryo following re-incubation. Dye is constrained towards the back of the eye (arrow). Pink line marks boundary between lens and OCL. L–N: Coronal sections through the embryo in J, K. L: DiI (red arrows) is in the central RPE and brain (red arrows) but absent from other eye domains. M: Higher magnification of the boxed area in L. DiI (red arrows) is distributed in the central RPE and in the brain. N: More temporally positioned section through the optic stalk. DiI is distributed from the central RPE through the optic stalk to the brain (red arrows). O–R: Optic vesicle labeling distributing in non-eye tissues. O, Q: Optic vesicle labeling in HH9 embryos. P, R: Distribution of dye in the CNS (arrows) following the labeling in O and Q respectively. No dye was distributed to the eye. S : Scheme summarizing dye distribution of OV dye labels into the central OC. Central RPE arose from the dorsal OV (yellow) and central NR from the posterior OV, adjacent to paraxial mesoderm (red). Proximal OV label distributed label to the CNS (grey). OV-optic vesicle, CNS-central nervous system, L-lens, NR-neural retina, RPE-retinal pigmented epithelium, A-anterior, P-posterior, Pr-proximal, Di-distal, D-dorsal, V-ventral, b-brain, L-lens. Scale bars = 100 µm.
    Figure Legend Snippet: Identification of Central Optic Cup Fields in the Optic Vesicle. A: Schematic dorsal view of an optic vesicle at HH9/10 summarizing non-distal OV zones. B: Dye targeted to the posterior OV (arrow). C–E: Coronal sections of B following re-incubation. C: Low magnification. D–E: Higher magnification showing dye distributed in the central neural retina (red arrows). Dye is excluded from the OCL and posterior RPE. F: Dorsal view with DiI at the distal OV (red arrow) and DiO proximal (green arrows). G–I: Coronal section through the embryo in F following reincubation. G: DiI is distributed to the OCL. The central limit of DiI distribution is indicated (red arrow). H: DiO is distributed through the central RPE. The peripheral limit of DiO distribution is indicated (green arrow). I: Composite of G/H. DiI (red) in the OCL and DiO (green) in the central RPE do not overlap. J: Dorsal view of dye label proximal in the OV (red arrow). K: The same embryo following re-incubation. Dye is constrained towards the back of the eye (arrow). Pink line marks boundary between lens and OCL. L–N: Coronal sections through the embryo in J, K. L: DiI (red arrows) is in the central RPE and brain (red arrows) but absent from other eye domains. M: Higher magnification of the boxed area in L. DiI (red arrows) is distributed in the central RPE and in the brain. N: More temporally positioned section through the optic stalk. DiI is distributed from the central RPE through the optic stalk to the brain (red arrows). O–R: Optic vesicle labeling distributing in non-eye tissues. O, Q: Optic vesicle labeling in HH9 embryos. P, R: Distribution of dye in the CNS (arrows) following the labeling in O and Q respectively. No dye was distributed to the eye. S : Scheme summarizing dye distribution of OV dye labels into the central OC. Central RPE arose from the dorsal OV (yellow) and central NR from the posterior OV, adjacent to paraxial mesoderm (red). Proximal OV label distributed label to the CNS (grey). OV-optic vesicle, CNS-central nervous system, L-lens, NR-neural retina, RPE-retinal pigmented epithelium, A-anterior, P-posterior, Pr-proximal, Di-distal, D-dorsal, V-ventral, b-brain, L-lens. Scale bars = 100 µm.

    Techniques Used: Incubation, Labeling

    9) Product Images from "Delineating the rules for structural adaptation of membrane-associated proteins to evolutionary changes in membrane lipidome"

    Article Title: Delineating the rules for structural adaptation of membrane-associated proteins to evolutionary changes in membrane lipidome

    Journal: bioRxiv

    doi: 10.1101/762146

    ( A ) Examples of the primary microscopy data used to calculate Ld/Lo values shown in Fig. 2B . Note differences in FAST DIO staining intensities between GUVs formed from S. pombe and S. japonicus phospholipids – this dye preferentially partitions to Ld membranes. Intensity calibration bars are included. Scale bar, 5 μm. ( B ) A plot describing the results of the GC-MS analysis of indicated 13 C-labeled FAs extracted from S. japonicus cells grown in the presence of the U- 13 C-glucose for 4 hours. 10 μM cerulenin was added when indicated. Shown are the means of percentages of 13 C-labeled fatty acids ± SD (n = 6). ( C ) Relative abundance of C10:0 in the wild type and three mutants in the mitochondrial FA synthesis pathway. Genotypes of mutants are indicated. ( D ) A line chart tracing relative absorbance of NADPH at 340 nm in protein fractions used for in vitro FA synthesis reactions shown in Fig. 2F . Malonyl-CoA was added at time 0. Graphs representing molecular species profiles for phosphatidylcholine (PC) ( E ), phosphatidylinositol (PI) ( F ), phosphatidylethanolamine (PE) ( G ) and phosphatidylserine (PS) ( H ) in S. pombe wild type and S. pombe fas s. j. cells. Phospholipids are specified by the total carbon atoms: total double bonds in acyls. Broken columns are used to show details at the bottom and the top of the scale. ( I ) A representative mass spectrum of the PI 28:1 molecular species from the S. pombe fas s. j. lipid extract indicating ions generated upon fragmentation. Graphs representing the abundance of the four main GPL classes (PC, PI, PE, and PS) ( J ) and other indicated membrane lipids ( K ) in S. pombe wild type and fas s. j. cells, presented as molecular percentages of membrane lipids. ( L ) Relative abundance of C10:0 in the wild type and acb1 mutants of indicated genotypes. ( C , L ) Shown are the means of relative C10:0 content ± SD (n = 6). p values derived from the unpaired parametric t-test. ( E - H, J-K ) Shown are the mean values ± SD ( n = 4 for the wild type and 5 for fas s. j. cells). p values derived from the unpaired parametric t-test.
    Figure Legend Snippet: ( A ) Examples of the primary microscopy data used to calculate Ld/Lo values shown in Fig. 2B . Note differences in FAST DIO staining intensities between GUVs formed from S. pombe and S. japonicus phospholipids – this dye preferentially partitions to Ld membranes. Intensity calibration bars are included. Scale bar, 5 μm. ( B ) A plot describing the results of the GC-MS analysis of indicated 13 C-labeled FAs extracted from S. japonicus cells grown in the presence of the U- 13 C-glucose for 4 hours. 10 μM cerulenin was added when indicated. Shown are the means of percentages of 13 C-labeled fatty acids ± SD (n = 6). ( C ) Relative abundance of C10:0 in the wild type and three mutants in the mitochondrial FA synthesis pathway. Genotypes of mutants are indicated. ( D ) A line chart tracing relative absorbance of NADPH at 340 nm in protein fractions used for in vitro FA synthesis reactions shown in Fig. 2F . Malonyl-CoA was added at time 0. Graphs representing molecular species profiles for phosphatidylcholine (PC) ( E ), phosphatidylinositol (PI) ( F ), phosphatidylethanolamine (PE) ( G ) and phosphatidylserine (PS) ( H ) in S. pombe wild type and S. pombe fas s. j. cells. Phospholipids are specified by the total carbon atoms: total double bonds in acyls. Broken columns are used to show details at the bottom and the top of the scale. ( I ) A representative mass spectrum of the PI 28:1 molecular species from the S. pombe fas s. j. lipid extract indicating ions generated upon fragmentation. Graphs representing the abundance of the four main GPL classes (PC, PI, PE, and PS) ( J ) and other indicated membrane lipids ( K ) in S. pombe wild type and fas s. j. cells, presented as molecular percentages of membrane lipids. ( L ) Relative abundance of C10:0 in the wild type and acb1 mutants of indicated genotypes. ( C , L ) Shown are the means of relative C10:0 content ± SD (n = 6). p values derived from the unpaired parametric t-test. ( E - H, J-K ) Shown are the mean values ± SD ( n = 4 for the wild type and 5 for fas s. j. cells). p values derived from the unpaired parametric t-test.

    Techniques Used: Microscopy, Staining, Gas Chromatography-Mass Spectrometry, Labeling, In Vitro, Generated, Derivative Assay

    10) Product Images from "Origin and early development of the chicken adenohypophysis"

    Article Title: Origin and early development of the chicken adenohypophysis

    Journal: Frontiers in Neuroanatomy

    doi: 10.3389/fnana.2015.00007

    Adenohypophyseal fate: analysis of the experimental cases RPA-27, RPA-02 and RPA-80. Initial median cell movements. (A) Map of all studied grafts or injections corresponding with partial adenohypophyseal fate. (B,D,F,H) Combined fluorescent and bright field images of representative HH4 chick embryos in which a graft (B) or either a DiI (D) or DiO injection (F,H ) was placed at the median non-neural ectodermal region. (C,C’) At stage HH11, the graft-derived cells were located in the rostromedian non-neural ectoderm, largely coinciding with the Fgf8 -negative Rathke’s pouch rudiment. (E,E’) The prospective Rathke’s pouch tissue, flipped over into external contact with the neural terminal wall, already has elongated to the level of the Shh -positive prospective hypothalamic basal plate. (F,G) In case RPA-80, the labeled non-neural cells of the ADH primordium clearly appeared at the medial head ectoderm, within the space that separates the bilateral Raldh3 -positive olfactory placodes, whose cells remained unlabeled. (I,J) Simple fluorescent and combined fluorescent and bright field images of the case illustrated in (H) , counterstained with Dlx5 whole-mount ISH, showing the elongated median labeled trace of ectodermal tissue extending from the preplacodal field (ppf) into the ventrally displaced ADH primordium, at stage HH6. Arrowheads in (I) mark the site of the original injection (see also Sanchez-Arrones et al., 2012 ). Scale bar: 250 μm in (B,C) ; 125 μm in (C’) .
    Figure Legend Snippet: Adenohypophyseal fate: analysis of the experimental cases RPA-27, RPA-02 and RPA-80. Initial median cell movements. (A) Map of all studied grafts or injections corresponding with partial adenohypophyseal fate. (B,D,F,H) Combined fluorescent and bright field images of representative HH4 chick embryos in which a graft (B) or either a DiI (D) or DiO injection (F,H ) was placed at the median non-neural ectodermal region. (C,C’) At stage HH11, the graft-derived cells were located in the rostromedian non-neural ectoderm, largely coinciding with the Fgf8 -negative Rathke’s pouch rudiment. (E,E’) The prospective Rathke’s pouch tissue, flipped over into external contact with the neural terminal wall, already has elongated to the level of the Shh -positive prospective hypothalamic basal plate. (F,G) In case RPA-80, the labeled non-neural cells of the ADH primordium clearly appeared at the medial head ectoderm, within the space that separates the bilateral Raldh3 -positive olfactory placodes, whose cells remained unlabeled. (I,J) Simple fluorescent and combined fluorescent and bright field images of the case illustrated in (H) , counterstained with Dlx5 whole-mount ISH, showing the elongated median labeled trace of ectodermal tissue extending from the preplacodal field (ppf) into the ventrally displaced ADH primordium, at stage HH6. Arrowheads in (I) mark the site of the original injection (see also Sanchez-Arrones et al., 2012 ). Scale bar: 250 μm in (B,C) ; 125 μm in (C’) .

    Techniques Used: Recombinase Polymerase Amplification, Injection, Derivative Assay, Labeling, In Situ Hybridization

    11) Product Images from "Sequence Variations and Protein Expression Levels of the Two Immune Evasion Proteins Gpm1 and Pra1 Influence Virulence of Clinical Candida albicans Isolates"

    Article Title: Sequence Variations and Protein Expression Levels of the Two Immune Evasion Proteins Gpm1 and Pra1 Influence Virulence of Clinical Candida albicans Isolates

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0113192

    The clinical C . albicans isolates attach to human endothelial cells. ( A ) J#7, the high Gpm1-, Pra1 expressing clinical isolate was co-cultivated with human endothelial cells (HUVEC) for 2.5 h at 37°C. After washing, yeast cells were stained by anti-cell wall serum and in combination with FITC labeled goat anti rabbit serum (green fluorescence). HUVEC cells were stained by DAPI (blue) and evaluated by laser scanning microscopy. C . albicans (green) either adhered or invaded the HUVEC cells (blue). ( B ) To quantify how Gpm1- and Pra1 surface levels influence the interaction of different clinical C . albicans isolates with HUVEC, overnight cultures of the selected clinical isolates and of the reference strain SC5314 (1 x 10 6 ) were washed in DPBS, then stained with calcofluor (1 μg/ml) for 30 min at 37°C. After washing the yeast cells were added to DiO (1:100) labeled HUVEC cells, which were kept in FCS (fetal calf serum) free DMEM medium. After co-cultivation (2.5 h), unbound Candida cells were removed by extensive washing, then HUVEC cells together with the adherent and invaded C . albicans cells were detached, washed again and the samples were evaluated by flow cytometry. The blot shown here is an example of one clinical isolate (J#7). DiO labeled HUVEC cells were identified as single positive cells (DiO + , Calcofluor - ) (panel I) . HUVEC cells with adherent and ingested yeast cells were identified as double positive cells (DiO + , Calcofluor + ) ( panel II ). ( C ) Comparison of the infection ability of different clinical C . albicans isolates. Different clinical C . abicans isolates, reference strain SC5314 and HUVEC cells were prepared and labeled as shown in (B). After co-cultivation (2.5 h), the percentage of infected HUVEC cells by different clinical C . albicans isolates were recorded by flow cytometry as double positive cells. The percentage of double positive HUVEC cells were used to indicate the infection ability of different C . albicans isolates. The mean values of each group, i.e. the low, medium or high Gpm1/Pra1 expressing isolates are indicated by the crossed circle in the middle of each group. The bars represent the mean values of three independent experiments ± S.D. To better quantify the adhesion and invasion ability of different clinical strains to human endothelial HUVEC cells, C . albicans isolates labeled with calcofluor were co-cultivated with DiO labeled HUVEC cells, afterwards the interaction of yeast and human cells were analyzed by flow cytometry. Unbound and loosely attached Candida cells were removed by washing. HUVEC cells that had no C . albicans attached were identified as single positive cells (Calcofluor - , DiO + ) ( Fig. 5 B , panel I). In contrast HUVEC cells with adherent and invaded Candida cells were identified as double positive cells (Calcofluor + and DiO + ) ( Fig. 5 B , panel II). Upon co-cultivation of the high expressing strain J#7, about 23% of the human endothelial cells had C . albicans infected.
    Figure Legend Snippet: The clinical C . albicans isolates attach to human endothelial cells. ( A ) J#7, the high Gpm1-, Pra1 expressing clinical isolate was co-cultivated with human endothelial cells (HUVEC) for 2.5 h at 37°C. After washing, yeast cells were stained by anti-cell wall serum and in combination with FITC labeled goat anti rabbit serum (green fluorescence). HUVEC cells were stained by DAPI (blue) and evaluated by laser scanning microscopy. C . albicans (green) either adhered or invaded the HUVEC cells (blue). ( B ) To quantify how Gpm1- and Pra1 surface levels influence the interaction of different clinical C . albicans isolates with HUVEC, overnight cultures of the selected clinical isolates and of the reference strain SC5314 (1 x 10 6 ) were washed in DPBS, then stained with calcofluor (1 μg/ml) for 30 min at 37°C. After washing the yeast cells were added to DiO (1:100) labeled HUVEC cells, which were kept in FCS (fetal calf serum) free DMEM medium. After co-cultivation (2.5 h), unbound Candida cells were removed by extensive washing, then HUVEC cells together with the adherent and invaded C . albicans cells were detached, washed again and the samples were evaluated by flow cytometry. The blot shown here is an example of one clinical isolate (J#7). DiO labeled HUVEC cells were identified as single positive cells (DiO + , Calcofluor - ) (panel I) . HUVEC cells with adherent and ingested yeast cells were identified as double positive cells (DiO + , Calcofluor + ) ( panel II ). ( C ) Comparison of the infection ability of different clinical C . albicans isolates. Different clinical C . abicans isolates, reference strain SC5314 and HUVEC cells were prepared and labeled as shown in (B). After co-cultivation (2.5 h), the percentage of infected HUVEC cells by different clinical C . albicans isolates were recorded by flow cytometry as double positive cells. The percentage of double positive HUVEC cells were used to indicate the infection ability of different C . albicans isolates. The mean values of each group, i.e. the low, medium or high Gpm1/Pra1 expressing isolates are indicated by the crossed circle in the middle of each group. The bars represent the mean values of three independent experiments ± S.D. To better quantify the adhesion and invasion ability of different clinical strains to human endothelial HUVEC cells, C . albicans isolates labeled with calcofluor were co-cultivated with DiO labeled HUVEC cells, afterwards the interaction of yeast and human cells were analyzed by flow cytometry. Unbound and loosely attached Candida cells were removed by washing. HUVEC cells that had no C . albicans attached were identified as single positive cells (Calcofluor - , DiO + ) ( Fig. 5 B , panel I). In contrast HUVEC cells with adherent and invaded Candida cells were identified as double positive cells (Calcofluor + and DiO + ) ( Fig. 5 B , panel II). Upon co-cultivation of the high expressing strain J#7, about 23% of the human endothelial cells had C . albicans infected.

    Techniques Used: Expressing, Staining, Labeling, Fluorescence, Laser-Scanning Microscopy, Flow Cytometry, Cytometry, Infection

    12) Product Images from "Mesenchymal stem cells differentially affect the invasion of distinct glioblastoma cell lines"

    Article Title: Mesenchymal stem cells differentially affect the invasion of distinct glioblastoma cell lines

    Journal: Oncotarget

    doi: 10.18632/oncotarget.16041

    Proliferation and invasion of U87 dsRED and U373 eGFP cells in the zebrafish embryo brain upon co-injection with DiO/DiI- labelled MSCs (A) Two days after zebrafish embryo fertilisation, U87 and U373 cells alone (left upper and lower panels) or mixed with fluorescently stained MSCs with DiO (green) in the case of U87 (right upper panel), and with DiI (red) in the case of U373 (right lower panel), were injected into the brains of the zebrafish embryos. Cell nuclei were stained with methyl green (magnification, 10×, green blue shapes; scale bar = 250 μm). (B) GBM cell proliferation was determined 24 h and 72 after the injections by confocal microscopy and quantified as relative fluorescence intensity of U373eGFP and U87dsRed labelled cells injected alone or with MSCs (DC). (C) Relative invasion of U87dsRed and U373eGFP cells injected alone, or with fluorescently stained MSCs (DC) was determined as described in Material and Methods, clearly showing increased U373eGFP invasiveness and reduced U87 invasiveness from co-culture xenografts. Thirty zebrafish embryos were used per group. Data are means ± SD. ** P
    Figure Legend Snippet: Proliferation and invasion of U87 dsRED and U373 eGFP cells in the zebrafish embryo brain upon co-injection with DiO/DiI- labelled MSCs (A) Two days after zebrafish embryo fertilisation, U87 and U373 cells alone (left upper and lower panels) or mixed with fluorescently stained MSCs with DiO (green) in the case of U87 (right upper panel), and with DiI (red) in the case of U373 (right lower panel), were injected into the brains of the zebrafish embryos. Cell nuclei were stained with methyl green (magnification, 10×, green blue shapes; scale bar = 250 μm). (B) GBM cell proliferation was determined 24 h and 72 after the injections by confocal microscopy and quantified as relative fluorescence intensity of U373eGFP and U87dsRed labelled cells injected alone or with MSCs (DC). (C) Relative invasion of U87dsRed and U373eGFP cells injected alone, or with fluorescently stained MSCs (DC) was determined as described in Material and Methods, clearly showing increased U373eGFP invasiveness and reduced U87 invasiveness from co-culture xenografts. Thirty zebrafish embryos were used per group. Data are means ± SD. ** P

    Techniques Used: Injection, Staining, Confocal Microscopy, Fluorescence, Co-Culture Assay

    Invasion of DiO/DiI-labelled MSCs and GBM (U87 dsRED, U373 eGFP) cells from spheroids upon treatment with selective inhibitors of cathepsin B (2 μM), MMP-9 (100 nM) and MMP-14 (10 μM) Generated monospheroids (MSCs, U87, U373) and mixed spheroids were incubated in laminin-coated wells and treated with protease inhibitors or control (0.1% DMSO). (A) Invasion (invasion distance/ spheroid diameter) of MSCs and U87 cells from spheroids in the presence of protease inhibitors or control medium, measured after 72 h. (B) Invasion of MSCs and U373 cells from spheroids in the presence of protease inhibitors or control medium after 72 h. Data are means ± SD. * P
    Figure Legend Snippet: Invasion of DiO/DiI-labelled MSCs and GBM (U87 dsRED, U373 eGFP) cells from spheroids upon treatment with selective inhibitors of cathepsin B (2 μM), MMP-9 (100 nM) and MMP-14 (10 μM) Generated monospheroids (MSCs, U87, U373) and mixed spheroids were incubated in laminin-coated wells and treated with protease inhibitors or control (0.1% DMSO). (A) Invasion (invasion distance/ spheroid diameter) of MSCs and U87 cells from spheroids in the presence of protease inhibitors or control medium, measured after 72 h. (B) Invasion of MSCs and U373 cells from spheroids in the presence of protease inhibitors or control medium after 72 h. Data are means ± SD. * P

    Techniques Used: Generated, Incubation

    Invasion of DiO/DiI-labelled MSCs, U87dsRed and U373eGFP cells from spheroids Spheroids of MSCs, GBM cells (monocultured) and MSC/GBM cells as direct co-cultures (DC) were prepared and embedded in laminin, collagen I and Matrigel. Invasion distance versus spheroid diameter (Invasion) was measured over a period of 4 days using a fluorescent inverted microscope. (A) Invasion of U87 cells (left) and U373 cells (right) from spheroids. (B) Invasion of MSCs from spheroids, as MSCs co-cultured with U87 cells (left) and U373 cells (right). (C) Representative images of MSCs and U87 and U373 cells invading from monocultures and MSC/GBM direct co-cultures (DC) after 2 days in collagen I (magnification, 40×). Scale bar = 200 μm. Data are means ± SD. * P
    Figure Legend Snippet: Invasion of DiO/DiI-labelled MSCs, U87dsRed and U373eGFP cells from spheroids Spheroids of MSCs, GBM cells (monocultured) and MSC/GBM cells as direct co-cultures (DC) were prepared and embedded in laminin, collagen I and Matrigel. Invasion distance versus spheroid diameter (Invasion) was measured over a period of 4 days using a fluorescent inverted microscope. (A) Invasion of U87 cells (left) and U373 cells (right) from spheroids. (B) Invasion of MSCs from spheroids, as MSCs co-cultured with U87 cells (left) and U373 cells (right). (C) Representative images of MSCs and U87 and U373 cells invading from monocultures and MSC/GBM direct co-cultures (DC) after 2 days in collagen I (magnification, 40×). Scale bar = 200 μm. Data are means ± SD. * P

    Techniques Used: Inverted Microscopy, Cell Culture

    13) Product Images from "Mutation of Vsx genes in zebrafish highlights the robustness of the retinal specification network"

    Article Title: Mutation of Vsx genes in zebrafish highlights the robustness of the retinal specification network

    Journal: bioRxiv

    doi: 10.1101/2022.01.20.477122

    vsx KO larvae show normal GCL retinotectal projections. a, b. 3-D reconstructions of confocal stacks from zebrafish larval eyes injected with either DiO (green) or DiI (red) to label retinal ganglion cells and their projections to the optic tectum in wildtype (a, n=6) and vsx KO (b, n=8) at 6dpf. Note that vsx KO larvae show aparently normal retinotectal projections.
    Figure Legend Snippet: vsx KO larvae show normal GCL retinotectal projections. a, b. 3-D reconstructions of confocal stacks from zebrafish larval eyes injected with either DiO (green) or DiI (red) to label retinal ganglion cells and their projections to the optic tectum in wildtype (a, n=6) and vsx KO (b, n=8) at 6dpf. Note that vsx KO larvae show aparently normal retinotectal projections.

    Techniques Used: Injection

    14) Product Images from "Galectin-3-Binding and Metastasis"

    Article Title: Galectin-3-Binding and Metastasis

    Journal: Methods in molecular biology (Clifton, N.J.)

    doi: 10.1007/978-1-61779-854-2_17

    Endothelial cell morphogenesis and interactions with H64 or P64 transfected BT-549 cells. Endothelial cells BAMEC and Galectin-3 variant H64 or P64 transfected BT-549 cells were prelabeled with DiO (green) and DiI (red) respectively. 50,000 cells were seeded in each chamber on top of gelled Matrigel. The 3-dimensional cultures were observed after 24 hr. a: BAMEC alone; b: BAMEC and BT-549 cells; c: BAMEC and BT-549 H64; d: BAMEC and BT-549 P64.
    Figure Legend Snippet: Endothelial cell morphogenesis and interactions with H64 or P64 transfected BT-549 cells. Endothelial cells BAMEC and Galectin-3 variant H64 or P64 transfected BT-549 cells were prelabeled with DiO (green) and DiI (red) respectively. 50,000 cells were seeded in each chamber on top of gelled Matrigel. The 3-dimensional cultures were observed after 24 hr. a: BAMEC alone; b: BAMEC and BT-549 cells; c: BAMEC and BT-549 H64; d: BAMEC and BT-549 P64.

    Techniques Used: Transfection, Variant Assay

    Cell migration using wound healing assay: Endothelial cells BAMEC and Galectin-3 variant H64 or P64 transfected BT-549 cells were prelabeled with DiO (green) and DiI (red) respectively and seeded in each chamber of cell culture insert. After 24 hr, the inserts were removed and cell migration was studied. a–a′: migration of BAMEC and BT-549-H64; b–b′: migration of BAMEC and BT-549-P64; a,b : 0 hr; a′b′: 24 hr.
    Figure Legend Snippet: Cell migration using wound healing assay: Endothelial cells BAMEC and Galectin-3 variant H64 or P64 transfected BT-549 cells were prelabeled with DiO (green) and DiI (red) respectively and seeded in each chamber of cell culture insert. After 24 hr, the inserts were removed and cell migration was studied. a–a′: migration of BAMEC and BT-549-H64; b–b′: migration of BAMEC and BT-549-P64; a,b : 0 hr; a′b′: 24 hr.

    Techniques Used: Migration, Wound Healing Assay, Variant Assay, Transfection, Cell Culture

    15) Product Images from "Glucocorticoid-induced loss of beneficial gut bacterial extracellular vesicles is associated with the pathogenesis of osteonecrosis"

    Article Title: Glucocorticoid-induced loss of beneficial gut bacterial extracellular vesicles is associated with the pathogenesis of osteonecrosis

    Journal: Science Advances

    doi: 10.1126/sciadv.abg8335

    L. animalis -EVs enter the femoral head and mitigate GC-induced ONFH. ( A ) Distribution of the DiR-labeled L. animalis -EVs in the femurs, tibias, and femoral heads of mice detected by ex vivo fluorescent imaging after oral administration for 3, 24, and 72 hours. Scale bar, 6 mm. ( B ) Quantification of the fluorescence intensity. n = 3 per group. ( C ) Distribution of the DiO-labeled L. animalis -EVs within the mouse femoral head after oral administration for 3 hours. Scale bars, 50 μm (white) or 20 μm (red). ( D ) Schematic diagram of the experimental design for testing the effects of oral treatment with L. animalis -EVs on the femoral heads of vehicle- or MPS-treated mice. ( E ) Detection of L. animalis -EVs in the mouse femoral heads using the antibodies targeting L. animalis -EVs ( L -EVs Ab). Scale bars, 50 μm (white) or 20 μm (red). ( F ) Quantification of the mean intensity for L -EV + areas. n = 3 per group. ( G to K ) μCT-reconstructed images of femoral heads (G) and quantification of Tb. BV/TV (H), Tb. Th (I), Tb. N (J), and Tb. Sp (K). Scale bar, 1 mm. n = 8 per group. ( L ) H E staining images of femoral heads. Scale bars, 200 μm (blue) or 50 μm (black). ( M to P ) Quantification of total vessel volume (M) and the numbers of CD31 + (N), OCN + (O), and TUNEL + (P) cells in femoral heads. n = 5 per group. * P
    Figure Legend Snippet: L. animalis -EVs enter the femoral head and mitigate GC-induced ONFH. ( A ) Distribution of the DiR-labeled L. animalis -EVs in the femurs, tibias, and femoral heads of mice detected by ex vivo fluorescent imaging after oral administration for 3, 24, and 72 hours. Scale bar, 6 mm. ( B ) Quantification of the fluorescence intensity. n = 3 per group. ( C ) Distribution of the DiO-labeled L. animalis -EVs within the mouse femoral head after oral administration for 3 hours. Scale bars, 50 μm (white) or 20 μm (red). ( D ) Schematic diagram of the experimental design for testing the effects of oral treatment with L. animalis -EVs on the femoral heads of vehicle- or MPS-treated mice. ( E ) Detection of L. animalis -EVs in the mouse femoral heads using the antibodies targeting L. animalis -EVs ( L -EVs Ab). Scale bars, 50 μm (white) or 20 μm (red). ( F ) Quantification of the mean intensity for L -EV + areas. n = 3 per group. ( G to K ) μCT-reconstructed images of femoral heads (G) and quantification of Tb. BV/TV (H), Tb. Th (I), Tb. N (J), and Tb. Sp (K). Scale bar, 1 mm. n = 8 per group. ( L ) H E staining images of femoral heads. Scale bars, 200 μm (blue) or 50 μm (black). ( M to P ) Quantification of total vessel volume (M) and the numbers of CD31 + (N), OCN + (O), and TUNEL + (P) cells in femoral heads. n = 5 per group. * P

    Techniques Used: Labeling, Mouse Assay, Ex Vivo, Imaging, Fluorescence, Staining, TUNEL Assay

    L. animalis -EVs directly promote angiogenesis, augment osteogenesis, and reduce cell apoptosis. ( A ) Morphology of L. animalis -EVs under a transmission electron microscope. Scale bar, 50 nm. ( B ) Diameter measurement of L. animalis -EVs by nanoparticle tracking analysis. ( C ) Quantification of vesicle numbers in 100 μg of L. animalis -EVs from five different batches (E1, E2, E3, E4, and E5) by nanoparticle tracking analysis. n = 3 per group. ( D ) Uptake of the DiO (green)– or DiI (red)–labeled L. animalis -EVs by HMECs, MLO-Y4, mouse preosteoblast MC3T3-E1 cells, and BMSCs. Scale bar, 10 μm. ( E ) Tube formation images of HMECs treated with vehicle, MPS, or MPS + different concentrations of L. animalis -EVs ( L -EVs). Scale bar, 200 μm. ( F and G ) Quantification of total loops (F) and total tube length (G). n = 3 per group. ( H ) Tube formation images of HMECs treated with vehicle, vehicle + L. animalis -EVs, vehicle + L. reuteri -EVs, MPS, MPS + L. animalis -EVs, or MPS + L. reuteri -EVs. Scale bar, 200 μm. ( I and J ) Quantification of total loops (I) and total tube length (J). n = 3 per group. ( K ) Alizarin red S (ARS) staining images of BMSCs with different treatments under osteogenic induction. Scale bar, 200 μm. ( L ) Quantification of the percentage of ARS + areas. n = 3 per group. ( M to P ) Cell counting kit-8 (CCK-8) analysis of HMECs (M), MLO-Y4 (N), MC3T3-E1 (O), and BMSCs (P) with different treatments. n = 3 per group. ( Q and R ) TUNEL staining images of different cell types receiving different treatments (Q) and quantification of the ratio of TUNEL + apoptotic cells (R). Scale bar, 20 μm. n = 3 per group. * P
    Figure Legend Snippet: L. animalis -EVs directly promote angiogenesis, augment osteogenesis, and reduce cell apoptosis. ( A ) Morphology of L. animalis -EVs under a transmission electron microscope. Scale bar, 50 nm. ( B ) Diameter measurement of L. animalis -EVs by nanoparticle tracking analysis. ( C ) Quantification of vesicle numbers in 100 μg of L. animalis -EVs from five different batches (E1, E2, E3, E4, and E5) by nanoparticle tracking analysis. n = 3 per group. ( D ) Uptake of the DiO (green)– or DiI (red)–labeled L. animalis -EVs by HMECs, MLO-Y4, mouse preosteoblast MC3T3-E1 cells, and BMSCs. Scale bar, 10 μm. ( E ) Tube formation images of HMECs treated with vehicle, MPS, or MPS + different concentrations of L. animalis -EVs ( L -EVs). Scale bar, 200 μm. ( F and G ) Quantification of total loops (F) and total tube length (G). n = 3 per group. ( H ) Tube formation images of HMECs treated with vehicle, vehicle + L. animalis -EVs, vehicle + L. reuteri -EVs, MPS, MPS + L. animalis -EVs, or MPS + L. reuteri -EVs. Scale bar, 200 μm. ( I and J ) Quantification of total loops (I) and total tube length (J). n = 3 per group. ( K ) Alizarin red S (ARS) staining images of BMSCs with different treatments under osteogenic induction. Scale bar, 200 μm. ( L ) Quantification of the percentage of ARS + areas. n = 3 per group. ( M to P ) Cell counting kit-8 (CCK-8) analysis of HMECs (M), MLO-Y4 (N), MC3T3-E1 (O), and BMSCs (P) with different treatments. n = 3 per group. ( Q and R ) TUNEL staining images of different cell types receiving different treatments (Q) and quantification of the ratio of TUNEL + apoptotic cells (R). Scale bar, 20 μm. n = 3 per group. * P

    Techniques Used: Transmission Assay, Microscopy, Labeling, Staining, Cell Counting, CCK-8 Assay, TUNEL Assay

    16) Product Images from "Synaptically-Competent Neurons Derived from Canine Embryonic Stem Cells by Lineage Selection with EGF and Noggin"

    Article Title: Synaptically-Competent Neurons Derived from Canine Embryonic Stem Cells by Lineage Selection with EGF and Noggin

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0019768

    Co-culture on primary astrocytes produces functional cESC-derived neurons. When cultured on confluent layers of astrocytes (A–C), canine stem cell-derived neurons grown with primary fetal neurons received functional inhibitory and/or excitatory synaptic inputs (E–H). (A, B) Confluent layers of canine fetal astrocytes were labeled with DiI membrane dye, which was not visible at 488 nm (inset), and used as substrates for co-culture. (C) Canine ESC-derived cells were labeled with DiO (arrow), which extended along the length of entire cell (arrowhead) within 24 hr, and not to neighbouring cells. (D) Peak number of cells receiving synaptic input was reached at week 4 (characterized in G) with nearly 75% of sampled cells receiving synaptic input. (E) Mean spontaneous excitatory postsynaptic currents in primary and cESC-derived neurons. Basic membrane characteristics and rise-times of synaptic currents were equivalent between cells. (F) Primary and cESC-derived neurons exhibited spontaneous inhibitory synaptic input after one week of co-culture. (G) At 3–4 weeks (shaded in D), primary fetal neurons exhibited predominantly inhibitory GABAergic synaptic activity, while cESC-derived neurons displayed more glutamatergic synaptic activity, almost solely in the presence of primary neurons. DAPI was used as nuclear counterstain (blue, A–C). Scale bar = 100 µm.
    Figure Legend Snippet: Co-culture on primary astrocytes produces functional cESC-derived neurons. When cultured on confluent layers of astrocytes (A–C), canine stem cell-derived neurons grown with primary fetal neurons received functional inhibitory and/or excitatory synaptic inputs (E–H). (A, B) Confluent layers of canine fetal astrocytes were labeled with DiI membrane dye, which was not visible at 488 nm (inset), and used as substrates for co-culture. (C) Canine ESC-derived cells were labeled with DiO (arrow), which extended along the length of entire cell (arrowhead) within 24 hr, and not to neighbouring cells. (D) Peak number of cells receiving synaptic input was reached at week 4 (characterized in G) with nearly 75% of sampled cells receiving synaptic input. (E) Mean spontaneous excitatory postsynaptic currents in primary and cESC-derived neurons. Basic membrane characteristics and rise-times of synaptic currents were equivalent between cells. (F) Primary and cESC-derived neurons exhibited spontaneous inhibitory synaptic input after one week of co-culture. (G) At 3–4 weeks (shaded in D), primary fetal neurons exhibited predominantly inhibitory GABAergic synaptic activity, while cESC-derived neurons displayed more glutamatergic synaptic activity, almost solely in the presence of primary neurons. DAPI was used as nuclear counterstain (blue, A–C). Scale bar = 100 µm.

    Techniques Used: Co-Culture Assay, Functional Assay, Derivative Assay, Cell Culture, Labeling, Activity Assay

    17) Product Images from "Severity of Infantile Nystagmus Syndrome-Like Ocular Motor Phenotype Is Linked to the Extent of the Underlying Optic Nerve Projection Defect in Zebrafish belladonna Mutant"

    Article Title: Severity of Infantile Nystagmus Syndrome-Like Ocular Motor Phenotype Is Linked to the Extent of the Underlying Optic Nerve Projection Defect in Zebrafish belladonna Mutant

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.4378-12.2012

    Phenotypes of bel mutant larvae. A–C , Maximum intensity projections of z -stacks showing projection of optic nerve fibers in bel mutant larvae. The RGC axons were labeled by injecting the green lipophilic tracer dye DiO in the right eye and the red lipophilic tracer dye DiI in the left eye. Here the projection from the right eye is shown. Anterior is up. Scale bar, 100 μm. Arrow points to side of injection. The wt larvae as well as some bel larvae have a complete contralateral projection ( A ). In some bel larvae a variable fraction of axons misprojects ipsilaterally leading to a bilateral projection ( B ). Finally, some bel larvae are achiasmatic having a complete ipsilateral projection ( C ). D–G , OKR sample traces of eye position (Se) during stimulation with horizontal moving gratings (Vs). Some bel larvae show a properly directed OKR ( E ). Other larvae show a reversal of the OKR ( F ). Finally, some larvae show a weaker OKR with strongly reduced eye velocity and unclear direction ( G ).
    Figure Legend Snippet: Phenotypes of bel mutant larvae. A–C , Maximum intensity projections of z -stacks showing projection of optic nerve fibers in bel mutant larvae. The RGC axons were labeled by injecting the green lipophilic tracer dye DiO in the right eye and the red lipophilic tracer dye DiI in the left eye. Here the projection from the right eye is shown. Anterior is up. Scale bar, 100 μm. Arrow points to side of injection. The wt larvae as well as some bel larvae have a complete contralateral projection ( A ). In some bel larvae a variable fraction of axons misprojects ipsilaterally leading to a bilateral projection ( B ). Finally, some bel larvae are achiasmatic having a complete ipsilateral projection ( C ). D–G , OKR sample traces of eye position (Se) during stimulation with horizontal moving gratings (Vs). Some bel larvae show a properly directed OKR ( E ). Other larvae show a reversal of the OKR ( F ). Finally, some larvae show a weaker OKR with strongly reduced eye velocity and unclear direction ( G ).

    Techniques Used: Mutagenesis, Labeling, Injection

    18) Product Images from "Epithelial to mesenchymal transition influences fibroblast phenotype in colorectal cancer by altering miR‐200 levels in extracellular vesicles). Epithelial to mesenchymal transition influences fibroblast phenotype in colorectal cancer by altering miR‐200 levels in extracellular vesicles"

    Article Title: Epithelial to mesenchymal transition influences fibroblast phenotype in colorectal cancer by altering miR‐200 levels in extracellular vesicles). Epithelial to mesenchymal transition influences fibroblast phenotype in colorectal cancer by altering miR‐200 levels in extracellular vesicles

    Journal: Journal of Extracellular Vesicles

    doi: 10.1002/jev2.12226

    Characterisation and transfer of CRC EVs. (a) Endosomal (Alix, TSG101) and tetraspanin (CD63, CD81) marker expression in HCT116 cells and EVs. The mitochondrial protein, cytochrome C, was used as a negative EV marker. Representative of three separate experiments. (b) Transmission electron micrograph of HCT116 EVs at 100,000× magnification. Scale bar represents 100 nm. Representative of three EV preparations. (c) Nanoparticle tracking analysis of HCT116 EVs from five separate videos, each 90s duration. (d) Visualisation of DiO‐labelled HCT116 EVs within MRC5 fibroblasts after 24 h conditioning. Phase contrast and fluorescence images are shown. Scale bar represents 50 μm. Representative of three experiments. (e) Detection of DiO‐labelled HCT116 EVs in MRC5 fibroblasts by flow cytometry. Representative histograms for control (DiO‐labelled medium) and EV‐conditioned fibroblasts from three experiments.
    Figure Legend Snippet: Characterisation and transfer of CRC EVs. (a) Endosomal (Alix, TSG101) and tetraspanin (CD63, CD81) marker expression in HCT116 cells and EVs. The mitochondrial protein, cytochrome C, was used as a negative EV marker. Representative of three separate experiments. (b) Transmission electron micrograph of HCT116 EVs at 100,000× magnification. Scale bar represents 100 nm. Representative of three EV preparations. (c) Nanoparticle tracking analysis of HCT116 EVs from five separate videos, each 90s duration. (d) Visualisation of DiO‐labelled HCT116 EVs within MRC5 fibroblasts after 24 h conditioning. Phase contrast and fluorescence images are shown. Scale bar represents 50 μm. Representative of three experiments. (e) Detection of DiO‐labelled HCT116 EVs in MRC5 fibroblasts by flow cytometry. Representative histograms for control (DiO‐labelled medium) and EV‐conditioned fibroblasts from three experiments.

    Techniques Used: Marker, Expressing, Transmission Assay, Fluorescence, Flow Cytometry

    19) Product Images from "Standing-wave-excited multiplanar fluorescence in a laser scanning microscope reveals 3D information on red blood cells"

    Article Title: Standing-wave-excited multiplanar fluorescence in a laser scanning microscope reveals 3D information on red blood cells

    Journal: Scientific Reports

    doi: 10.1038/srep07359

    Precise contour-mapping of the red blood cell membrane. Fringes in red blood cell ghosts ( a ) and intact red blood cells ( b , d , e ) from multiplanar excitation of fluorescence at the standing-wave antinodes. The cells were stained with the membrane-specific dyes DiI ( a and b ), DiO ( d ) or DiI(5) ( e ), and mounted on top of a mirror using 4% BSA in PBS (n = 1.34) to match the average refractive index of the membrane. Using an excitation wavelength of 488 nm for DiO, the axial resolution from the full width at half maximum of the high-intensity excitation light at the antinodes is λ /4 n ≈ 90 nm. Control specimens of red blood cell ghosts ( c ) and intact red blood cells ( f ) mounted on ordinary non-reflective glass microscope slides emit fluorescence only at the outline of the cell. Scale bar = 5 μ m.
    Figure Legend Snippet: Precise contour-mapping of the red blood cell membrane. Fringes in red blood cell ghosts ( a ) and intact red blood cells ( b , d , e ) from multiplanar excitation of fluorescence at the standing-wave antinodes. The cells were stained with the membrane-specific dyes DiI ( a and b ), DiO ( d ) or DiI(5) ( e ), and mounted on top of a mirror using 4% BSA in PBS (n = 1.34) to match the average refractive index of the membrane. Using an excitation wavelength of 488 nm for DiO, the axial resolution from the full width at half maximum of the high-intensity excitation light at the antinodes is λ /4 n ≈ 90 nm. Control specimens of red blood cell ghosts ( c ) and intact red blood cells ( f ) mounted on ordinary non-reflective glass microscope slides emit fluorescence only at the outline of the cell. Scale bar = 5 μ m.

    Techniques Used: Fluorescence, Staining, Microscopy

    20) Product Images from "Dual mode of paraxial mesoderm formation during chick gastrulation"

    Article Title: Dual mode of paraxial mesoderm formation during chick gastrulation

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.0610997104

    Medial and lateral somites are produced at different times by the anterior primitive streak. ( a ) Schematic representation of the DiI-/DiO-labeling procedure. ( b ) Stage 4+HH chicken embryo immediately after DiI/DiO labeling showing the position of labeled
    Figure Legend Snippet: Medial and lateral somites are produced at different times by the anterior primitive streak. ( a ) Schematic representation of the DiI-/DiO-labeling procedure. ( b ) Stage 4+HH chicken embryo immediately after DiI/DiO labeling showing the position of labeled

    Techniques Used: Produced, Labeling

    21) Product Images from "Fluorophore Exchange Kinetics in Block Copolymer Micelles with Varying Solvent-Fluorophore and Solvent-Polymer Interactions"

    Article Title: Fluorophore Exchange Kinetics in Block Copolymer Micelles with Varying Solvent-Fluorophore and Solvent-Polymer Interactions

    Journal: Soft matter

    doi: 10.1039/c6sm00297h

    Fluorescence emission spectra (at an excitation wavelength of 450 nm) are shown for micelle solutions containing a) 5 vol% THF, b) 10 vol% THF and c) 20 vol% THF. The spectra for micelles containing either DiO or DiI at t=0 are indicated by solid blue and green curves, respectively. The pre-mixed micelle solution (containing both fluorophores) at t=0 is indicated by a solid red curve. Spectra obtained after mixing the DiO and DiI-containing micelle samples (i.e. post-mixed samples) are shown as dashed curves (time points provided in the legend).
    Figure Legend Snippet: Fluorescence emission spectra (at an excitation wavelength of 450 nm) are shown for micelle solutions containing a) 5 vol% THF, b) 10 vol% THF and c) 20 vol% THF. The spectra for micelles containing either DiO or DiI at t=0 are indicated by solid blue and green curves, respectively. The pre-mixed micelle solution (containing both fluorophores) at t=0 is indicated by a solid red curve. Spectra obtained after mixing the DiO and DiI-containing micelle samples (i.e. post-mixed samples) are shown as dashed curves (time points provided in the legend).

    Techniques Used: Fluorescence

    22) Product Images from "Release of hydrophobic molecules from polymer micelles into cell membranes revealed by Förster resonance energy transfer imaging"

    Article Title: Release of hydrophobic molecules from polymer micelles into cell membranes revealed by Förster resonance energy transfer imaging

    Journal:

    doi: 10.1073/pnas.0707046105

    PEG-PDLLA FRET micelles. ( A ) Diagram of a FRET micelle prepared with 0.75% DiO and 0.75% DiI at 2 mg/ml polymer concentration. ( B ) Spectra of micelles diluted by 10× water (red curve) and 10× acetone (green curve), respectively.
    Figure Legend Snippet: PEG-PDLLA FRET micelles. ( A ) Diagram of a FRET micelle prepared with 0.75% DiO and 0.75% DiI at 2 mg/ml polymer concentration. ( B ) Spectra of micelles diluted by 10× water (red curve) and 10× acetone (green curve), respectively.

    Techniques Used: Concentration Assay

    Transfer of core-loaded probes to model membranes. ( A and B ) Fluorescence images of core-loaded DiO ( A ) and DiI ( B ) transferred to the DOPC supported bilayer. I DiI /( I DiI + I DiO ) = 0.37. ( C and D ) Fluorescence images of DiO ( C ) and DiI ( D ) FRET micelles
    Figure Legend Snippet: Transfer of core-loaded probes to model membranes. ( A and B ) Fluorescence images of core-loaded DiO ( A ) and DiI ( B ) transferred to the DOPC supported bilayer. I DiI /( I DiI + I DiO ) = 0.37. ( C and D ) Fluorescence images of DiO ( C ) and DiI ( D ) FRET micelles

    Techniques Used: Fluorescence

    23) Product Images from "Cooperative Astrocyte and Dendritic Spine Dynamics at Hippocampal Excitatory Synapses"

    Article Title: Cooperative Astrocyte and Dendritic Spine Dynamics at Hippocampal Excitatory Synapses

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.1302-06.2006

    Complex neuron–glial interactions in the hippocampal CA1 neuropil. a–c , Confocal maximum projections of a DiI-labeled apical dendrite from a CA1 pyramidal cell ( a , red) extending through a DiO-labeled astrocyte domain ( b , green) in a fixed slice of the adult hippocampus. Areas of overlap show the complexity of neuron–glial interactions. d–f , Confocal maximum projections of a dendrite from an SFV PD RFPf-infected CA1 pyramidal neuron ( d , red) projecting through an SFV A7 EGFPf-infected astrocyte domain ( e , green) after 1 week in vitro . g , h , Three-dimensional reconstructions of the boxed areas in f , demonstrating the complex interplay between astrocytic processes and dendritic spines. Inset in h shows the neuron–glial association rotated 90°. Arrowheads show examples of astrocytic processes extending toward spines. Scale bars, 5 μm.
    Figure Legend Snippet: Complex neuron–glial interactions in the hippocampal CA1 neuropil. a–c , Confocal maximum projections of a DiI-labeled apical dendrite from a CA1 pyramidal cell ( a , red) extending through a DiO-labeled astrocyte domain ( b , green) in a fixed slice of the adult hippocampus. Areas of overlap show the complexity of neuron–glial interactions. d–f , Confocal maximum projections of a dendrite from an SFV PD RFPf-infected CA1 pyramidal neuron ( d , red) projecting through an SFV A7 EGFPf-infected astrocyte domain ( e , green) after 1 week in vitro . g , h , Three-dimensional reconstructions of the boxed areas in f , demonstrating the complex interplay between astrocytic processes and dendritic spines. Inset in h shows the neuron–glial association rotated 90°. Arrowheads show examples of astrocytic processes extending toward spines. Scale bars, 5 μm.

    Techniques Used: Labeling, Infection, In Vitro

    Astrocytic elaboration and domain spacing in area CA1 of the adult mouse hippocampus and in organotypic slice culture. a–c , Diolistic labeling of protoplasmic astrocytes in fixed hippocampal tissue. Maximum projections of confocal images of neighboring astrocytes labeled with DiI ( a , red) and DiO ( b , green) reveals their distinct domain spacing. d–f , Astrocytes in organotypic hippocampal slice culture also occupy distinct territories. Maximum projections of confocal images of adjacent astrocytes expressing RFPf ( d , red) and EGFPf ( e , green) after 1 week in vitro . The dashed lines in c and f indicate the boundary between neighboring astrocytes. Scale bars, 10 μm.
    Figure Legend Snippet: Astrocytic elaboration and domain spacing in area CA1 of the adult mouse hippocampus and in organotypic slice culture. a–c , Diolistic labeling of protoplasmic astrocytes in fixed hippocampal tissue. Maximum projections of confocal images of neighboring astrocytes labeled with DiI ( a , red) and DiO ( b , green) reveals their distinct domain spacing. d–f , Astrocytes in organotypic hippocampal slice culture also occupy distinct territories. Maximum projections of confocal images of adjacent astrocytes expressing RFPf ( d , red) and EGFPf ( e , green) after 1 week in vitro . The dashed lines in c and f indicate the boundary between neighboring astrocytes. Scale bars, 10 μm.

    Techniques Used: Labeling, Expressing, In Vitro

    24) Product Images from "Eliminating the original cargos of glioblastoma cell-derived small extracellular vesicles for efficient drug delivery to glioblastoma with improved biosafety"

    Article Title: Eliminating the original cargos of glioblastoma cell-derived small extracellular vesicles for efficient drug delivery to glioblastoma with improved biosafety

    Journal: Bioactive Materials

    doi: 10.1016/j.bioactmat.2022.02.013

    Evaluation of targeting and accumulating ability of GBM-sEVs after saponin treatment. (A) CD63, CD9 and ITGB1 detected by WB. (B) Representative histogram of DiO fluorescence in U87 and U251 cells. (C and D) Confocal fluorescence microscopic images of U87 and U251 cells. Scale bar: 10 μm. (E) In vivo fluorescence images of the orthotopic brain tumor model by an IVIS Spectrum/CT imaging system (upper). Statistical analysis of the DiR fluorescence in vivo (lower). (F) Fluorescence images of the main organs of the orthotopic brain tumor bearing mice were captured 24 h after DiR-labeled sEVs were intravenously injected (upper). Statistical analysis of the DiR fluorescence of the main organs (lower). * P
    Figure Legend Snippet: Evaluation of targeting and accumulating ability of GBM-sEVs after saponin treatment. (A) CD63, CD9 and ITGB1 detected by WB. (B) Representative histogram of DiO fluorescence in U87 and U251 cells. (C and D) Confocal fluorescence microscopic images of U87 and U251 cells. Scale bar: 10 μm. (E) In vivo fluorescence images of the orthotopic brain tumor model by an IVIS Spectrum/CT imaging system (upper). Statistical analysis of the DiR fluorescence in vivo (lower). (F) Fluorescence images of the main organs of the orthotopic brain tumor bearing mice were captured 24 h after DiR-labeled sEVs were intravenously injected (upper). Statistical analysis of the DiR fluorescence of the main organs (lower). * P

    Techniques Used: Western Blot, Fluorescence, In Vivo, Imaging, Mouse Assay, Labeling, Injection

    25) Product Images from "The Role of the Immunological Synapse in Differential Effects of APC Subsets in Alloimmunization to Fresh, Non-stored RBCs"

    Article Title: The Role of the Immunological Synapse in Differential Effects of APC Subsets in Alloimmunization to Fresh, Non-stored RBCs

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.02200

    RBC clearance rates are similar between baseline and inflamed conditions. Recipient B6 mice were treated with 200 ug of poly (I:C) or control PBS and subsequently transfused with a 100 uL of 1:1 mixture of DiI+ B6 and DiO+ HOD RBCs. Whole blood was collected at multiple time points and (A) the ratio of DiO+ HOD RBCs to DiI+ B6 RBCs was determined to assess allogeneic RBC survival and (B) the overall percentage of DiO+ HOD RBCs was determined. Lines: poly (I:C) are red circles, PBS are blue squares. This experiment was repeated 3 times with 3–5 mice per group with similar results. A representative experiment is shown.
    Figure Legend Snippet: RBC clearance rates are similar between baseline and inflamed conditions. Recipient B6 mice were treated with 200 ug of poly (I:C) or control PBS and subsequently transfused with a 100 uL of 1:1 mixture of DiI+ B6 and DiO+ HOD RBCs. Whole blood was collected at multiple time points and (A) the ratio of DiO+ HOD RBCs to DiI+ B6 RBCs was determined to assess allogeneic RBC survival and (B) the overall percentage of DiO+ HOD RBCs was determined. Lines: poly (I:C) are red circles, PBS are blue squares. This experiment was repeated 3 times with 3–5 mice per group with similar results. A representative experiment is shown.

    Techniques Used: Mouse Assay

    Poly (I:C) leads to increased RBC consumption and upregulation of MHCII and CD86 expression. Recipient B6 mice were treated with poly (I:C) or control PBS and subsequently transfused with 100 uL of packed, leukoreduced, DiO-labeled HOD RBCs. At multiple time points, spleens were harvested, collagenase digested, and stained with antibodies to delineate APC subsets. (A) Total splenocyte counts were calculated and (B) the percent of DiO+ leukocytes was determined. The MFI of (C) DiO, (D) MHCII, and (E) CD86 was assessed for individual APC subsets. These experiments were repeated 3 times with 3 mice per group with similar results. A representative experiment is shown. PBS treated animals are shown with an open bar and poly (I:C) treatment is shown with a solid black bar. For analysis, T cells, B cells, and RBCs were excluded from total live leukocytes by gating out cells positive for Thy1.2, CD19, NK1.1, CD49b, and TER119. The following phenotypes were used to delineate APC subsets: RPMs: CD11c −/lo CD11b −/lo F4/80 + ; CD8+ DCs: CD11c hi CD11b − CD8 + ; CD11b+ DCs: CD11c hi CD11b + CD8 − ; pDCs: PDCA1 + CD11c int Ly6C hi ; inflammatory monocytes: CD11c −/lo CD11b + Ly6G − Ly6C hi CD115 + ; resident monocytes: CD11c −/lo CD11b + Ly6G − Ly6C lo CD115 − ; neutrophils: CD11c −/lo CD11b + Ly6G + and a high side scatter. For significance, **** p ≤ 0.0001, *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05.
    Figure Legend Snippet: Poly (I:C) leads to increased RBC consumption and upregulation of MHCII and CD86 expression. Recipient B6 mice were treated with poly (I:C) or control PBS and subsequently transfused with 100 uL of packed, leukoreduced, DiO-labeled HOD RBCs. At multiple time points, spleens were harvested, collagenase digested, and stained with antibodies to delineate APC subsets. (A) Total splenocyte counts were calculated and (B) the percent of DiO+ leukocytes was determined. The MFI of (C) DiO, (D) MHCII, and (E) CD86 was assessed for individual APC subsets. These experiments were repeated 3 times with 3 mice per group with similar results. A representative experiment is shown. PBS treated animals are shown with an open bar and poly (I:C) treatment is shown with a solid black bar. For analysis, T cells, B cells, and RBCs were excluded from total live leukocytes by gating out cells positive for Thy1.2, CD19, NK1.1, CD49b, and TER119. The following phenotypes were used to delineate APC subsets: RPMs: CD11c −/lo CD11b −/lo F4/80 + ; CD8+ DCs: CD11c hi CD11b − CD8 + ; CD11b+ DCs: CD11c hi CD11b + CD8 − ; pDCs: PDCA1 + CD11c int Ly6C hi ; inflammatory monocytes: CD11c −/lo CD11b + Ly6G − Ly6C hi CD115 + ; resident monocytes: CD11c −/lo CD11b + Ly6G − Ly6C lo CD115 − ; neutrophils: CD11c −/lo CD11b + Ly6G + and a high side scatter. For significance, **** p ≤ 0.0001, *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05.

    Techniques Used: Expressing, Mouse Assay, Labeling, Staining

    CD8+ and CD11b+ DCs promote proliferation and immune synapses with HOD RBC-specific T cells. Recipient B6 mice were treated with 200 ug of poly (I:C) or control PBS and subsequently transfused with 100 uL of leukoreduced, packed, DiO-labeled HOD RBCs. Spleens were harvested 18–24 h post transfusion, collagenase digested, and stained with antibodies to delineate individual APC populations. DiO+ APCs were sorted and co-cultured at a 10:1 ratio with CD4 enriched OTII T cells labeled with CFSE-FR. (A) Co-cultured cells were harvested after 3 days and CFSE-FR dilution was assessed in Va2+Vb5.1/5.2+CD4+Thy1.1+ OTII T cells. Lines: pDCs (green), CD11b+ DCs (red), CD8+ DCs (blue), RPM (purple), PMA/ionomycin (black), and media alone (gray). In separate experiments, co-cultured cells were harvested after 2 days and stained with antibodies to identify APCs, OTII T cells, and the immunological synapse. APCs were identified as MHCII+ (I-A b +) and either CD11c+ (for DCs) or F4/80+ (for RPMs) whereas OTIIs were defined as Va2+CD4+Thy1.1+. The immunological synapse was determined by co-expression of Va2, CD4, MHCII, and CD18 (also known as LFA-1). (B) The number and frequency of immunological synapses was determined and (C) representative images shown. These experiments were repeated 3 times with similar results; a representative experiment is shown.
    Figure Legend Snippet: CD8+ and CD11b+ DCs promote proliferation and immune synapses with HOD RBC-specific T cells. Recipient B6 mice were treated with 200 ug of poly (I:C) or control PBS and subsequently transfused with 100 uL of leukoreduced, packed, DiO-labeled HOD RBCs. Spleens were harvested 18–24 h post transfusion, collagenase digested, and stained with antibodies to delineate individual APC populations. DiO+ APCs were sorted and co-cultured at a 10:1 ratio with CD4 enriched OTII T cells labeled with CFSE-FR. (A) Co-cultured cells were harvested after 3 days and CFSE-FR dilution was assessed in Va2+Vb5.1/5.2+CD4+Thy1.1+ OTII T cells. Lines: pDCs (green), CD11b+ DCs (red), CD8+ DCs (blue), RPM (purple), PMA/ionomycin (black), and media alone (gray). In separate experiments, co-cultured cells were harvested after 2 days and stained with antibodies to identify APCs, OTII T cells, and the immunological synapse. APCs were identified as MHCII+ (I-A b +) and either CD11c+ (for DCs) or F4/80+ (for RPMs) whereas OTIIs were defined as Va2+CD4+Thy1.1+. The immunological synapse was determined by co-expression of Va2, CD4, MHCII, and CD18 (also known as LFA-1). (B) The number and frequency of immunological synapses was determined and (C) representative images shown. These experiments were repeated 3 times with similar results; a representative experiment is shown.

    Techniques Used: Mouse Assay, Labeling, Staining, Cell Culture, Expressing

    26) Product Images from "Evolutionarily conserved morphogenetic movements at the vertebrate head-trunk interface coordinate the transport and assembly of hypopharyngeal structures"

    Article Title: Evolutionarily conserved morphogenetic movements at the vertebrate head-trunk interface coordinate the transport and assembly of hypopharyngeal structures

    Journal: Developmental Biology

    doi: 10.1016/j.ydbio.2014.03.003

    DiI/DiO mapping of HMP versus lateral mesoderm cell movements. Dorsal or lateral views of chicken embryos as in Fig. 5 , with somite 3 (s3) labelled with DiI and the lateral mesoderm next to somite 1 (lm1; A–D), next to somite 2 (lm2; E–H) or next to somite 3 (lm3; I–L) labelled with DiO. The lateral mesoderm cell groups begin their morphogenetic movements before the deepithelialisation of TMP, with cells originally situated next to somite 1 (C, D; arrowhead) leading the TMP (C, D; arrow). The lateral mesodermal cell group originally found next to somite 2 split into two streams (G, H; arrowheads); the TMP are embedded in the rostral of these two streams (H, arrow). Lateral mesoderm originating from a position next to somite 3 are already displaced ventrolaterally when the TMP begin to head towards the floor of the pharyngeal arches (K, L). Abbreviations: ma, mandibular arch and ov, otic vesicle.
    Figure Legend Snippet: DiI/DiO mapping of HMP versus lateral mesoderm cell movements. Dorsal or lateral views of chicken embryos as in Fig. 5 , with somite 3 (s3) labelled with DiI and the lateral mesoderm next to somite 1 (lm1; A–D), next to somite 2 (lm2; E–H) or next to somite 3 (lm3; I–L) labelled with DiO. The lateral mesoderm cell groups begin their morphogenetic movements before the deepithelialisation of TMP, with cells originally situated next to somite 1 (C, D; arrowhead) leading the TMP (C, D; arrow). The lateral mesodermal cell group originally found next to somite 2 split into two streams (G, H; arrowheads); the TMP are embedded in the rostral of these two streams (H, arrow). Lateral mesoderm originating from a position next to somite 3 are already displaced ventrolaterally when the TMP begin to head towards the floor of the pharyngeal arches (K, L). Abbreviations: ma, mandibular arch and ov, otic vesicle.

    Techniques Used:

    DiI/DiO mapping of occipital neural crest cell versus lateral mesoderm cell movements. Dorsal or lateral views of chicken embryos as in Fig. 5 . (A–D) Tracing of neural crest cells (ncc). (A) Labelling of the neural folds overlying somites 1, 3, and 5 with DiI and DiO at HH8. (B) At HH12, nnc from the levels of somites 1 and 3 are actively migrating laterally; cells from the level of somite 5 have started their migration. (C, D) At HH16-18, ncc from somite level 1 fill the caudal pharyngeal arches, ncc from the level of somite 3 navigate around the most caudal arch (arrows), ncc from somite level 5 remain caudal to the ncc population from somite level 3 (open arrowhead). (E–H) Tracing of ncc originating from somite levels 3–4 with DiI and lateral mesoderm from somite 1 levels with DiO. (E) Labelling of cells at HH8-10. (F) At HH12, the lateral mesodermal cells have moved laterally–ventrally (arrowhead), the ncc are following behind (arrow). (G, H) At HH16-18, the lateral mesodermal cells move rostrally along the floor of the pharynx (arrowheads), closely followed by the ncc (arrowheads). Abbreviations: ma, mandibular arch and ov, otic vesicle.
    Figure Legend Snippet: DiI/DiO mapping of occipital neural crest cell versus lateral mesoderm cell movements. Dorsal or lateral views of chicken embryos as in Fig. 5 . (A–D) Tracing of neural crest cells (ncc). (A) Labelling of the neural folds overlying somites 1, 3, and 5 with DiI and DiO at HH8. (B) At HH12, nnc from the levels of somites 1 and 3 are actively migrating laterally; cells from the level of somite 5 have started their migration. (C, D) At HH16-18, ncc from somite level 1 fill the caudal pharyngeal arches, ncc from the level of somite 3 navigate around the most caudal arch (arrows), ncc from somite level 5 remain caudal to the ncc population from somite level 3 (open arrowhead). (E–H) Tracing of ncc originating from somite levels 3–4 with DiI and lateral mesoderm from somite 1 levels with DiO. (E) Labelling of cells at HH8-10. (F) At HH12, the lateral mesodermal cells have moved laterally–ventrally (arrowhead), the ncc are following behind (arrow). (G, H) At HH16-18, the lateral mesodermal cells move rostrally along the floor of the pharynx (arrowheads), closely followed by the ncc (arrowheads). Abbreviations: ma, mandibular arch and ov, otic vesicle.

    Techniques Used: Migration

    DiI/DiO labelling of hypobranchial/hypoglossal muscle precursors (HMP). Dorsal (A, B) or lateral (C–E) views of chicken embryos, rostral to the top. The occipital somites number 2 and 4 (s2, s4), both know to contribute to the hypobranchial/hypoglossal musculature, were labelled with DiI (red) and DiO (green) at HH8+ to HH10, and the position of these cells was recorded at the time points indicated on top of the panel. The labelled cells all collected in a single point at HH17-18 and travelled first ventrolaterally, then rostrally in a single stream. Abbreviations: ma, mandibular arch and ov, otic vesicle.
    Figure Legend Snippet: DiI/DiO labelling of hypobranchial/hypoglossal muscle precursors (HMP). Dorsal (A, B) or lateral (C–E) views of chicken embryos, rostral to the top. The occipital somites number 2 and 4 (s2, s4), both know to contribute to the hypobranchial/hypoglossal musculature, were labelled with DiI (red) and DiO (green) at HH8+ to HH10, and the position of these cells was recorded at the time points indicated on top of the panel. The labelled cells all collected in a single point at HH17-18 and travelled first ventrolaterally, then rostrally in a single stream. Abbreviations: ma, mandibular arch and ov, otic vesicle.

    Techniques Used:

    DiI/DiO labelling of the occipital lateral mesoderm and ectoderm reveals extensive cell movements. Dorsal (A, B) or lateral (C–E) views of the chicken head–neck interface at the stages indicated on top of the panel, rostral to the top. In each, the lateral mesoderm and overlying ectoderm next to somites 1, 3, 5, 7, and 9/10 (lm+ect 1, 3, 5, 7, and 9/10) had been labelled at stages HH8+ to HH10− with DiI (red) and DiO (green) as shown in (A). As development proceeded, the labelled cell groups became compressed rostrocaudally and stretched mediolaterally. Cell groups next to somite 1 (arrowhead) took the circumpharyngeal route, eventually extending rostrally along the floor of the arches and towards the mandibular arch; cells groups labelled next to somites 3–10 spread laterally–caudally. The cell movements matched the extension of Alx 4 and Wnt 6 expression shown in Fig. 1 . Abbreviations: ma, mandibular arch and ov, otic vesicle.
    Figure Legend Snippet: DiI/DiO labelling of the occipital lateral mesoderm and ectoderm reveals extensive cell movements. Dorsal (A, B) or lateral (C–E) views of the chicken head–neck interface at the stages indicated on top of the panel, rostral to the top. In each, the lateral mesoderm and overlying ectoderm next to somites 1, 3, 5, 7, and 9/10 (lm+ect 1, 3, 5, 7, and 9/10) had been labelled at stages HH8+ to HH10− with DiI (red) and DiO (green) as shown in (A). As development proceeded, the labelled cell groups became compressed rostrocaudally and stretched mediolaterally. Cell groups next to somite 1 (arrowhead) took the circumpharyngeal route, eventually extending rostrally along the floor of the arches and towards the mandibular arch; cells groups labelled next to somites 3–10 spread laterally–caudally. The cell movements matched the extension of Alx 4 and Wnt 6 expression shown in Fig. 1 . Abbreviations: ma, mandibular arch and ov, otic vesicle.

    Techniques Used: Expressing

    Position of occipital cells revealed by sections. (A) Lateral view of a HH19 chicken embryo stained for the expression of Pax 3. (B–F) Sections of HH19 embryos, stained for the markers indicated, along the frontal sectional plane denoted in (A); dorsal to the left. (G, H) Corresponding sections of embryos that at HH9-10 had been DiO-injected into the lateral mesoderm and ectoderm next to somite 1 (lm1) and DiI-injected into somite 3 (G, s3) or the neural crest at the level of somites 3 and 4 (ncc3/4). Abbreviations: dm, somitic dermomyotome; ect, surface ectoderm; end, endoderm; hy, hyoid arch; lm, lateral mesoderm; ma, mandibular arch; ncc, neural crest cells; nt, neural tube; ov, otic vesicle; splpl, splanchopleure; and V, trigeminal ganglion.
    Figure Legend Snippet: Position of occipital cells revealed by sections. (A) Lateral view of a HH19 chicken embryo stained for the expression of Pax 3. (B–F) Sections of HH19 embryos, stained for the markers indicated, along the frontal sectional plane denoted in (A); dorsal to the left. (G, H) Corresponding sections of embryos that at HH9-10 had been DiO-injected into the lateral mesoderm and ectoderm next to somite 1 (lm1) and DiI-injected into somite 3 (G, s3) or the neural crest at the level of somites 3 and 4 (ncc3/4). Abbreviations: dm, somitic dermomyotome; ect, surface ectoderm; end, endoderm; hy, hyoid arch; lm, lateral mesoderm; ma, mandibular arch; ncc, neural crest cells; nt, neural tube; ov, otic vesicle; splpl, splanchopleure; and V, trigeminal ganglion.

    Techniques Used: Staining, Expressing, Injection

    27) Product Images from "The Role of the Immunological Synapse in Differential Effects of APC Subsets in Alloimmunization to Fresh, Non-stored RBCs"

    Article Title: The Role of the Immunological Synapse in Differential Effects of APC Subsets in Alloimmunization to Fresh, Non-stored RBCs

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.02200

    RBC clearance rates are similar between baseline and inflamed conditions. Recipient B6 mice were treated with 200 ug of poly (I:C) or control PBS and subsequently transfused with a 100 uL of 1:1 mixture of DiI+ B6 and DiO+ HOD RBCs. Whole blood was collected at multiple time points and (A) the ratio of DiO+ HOD RBCs to DiI+ B6 RBCs was determined to assess allogeneic RBC survival and (B) the overall percentage of DiO+ HOD RBCs was determined. Lines: poly (I:C) are red circles, PBS are blue squares. This experiment was repeated 3 times with 3–5 mice per group with similar results. A representative experiment is shown.
    Figure Legend Snippet: RBC clearance rates are similar between baseline and inflamed conditions. Recipient B6 mice were treated with 200 ug of poly (I:C) or control PBS and subsequently transfused with a 100 uL of 1:1 mixture of DiI+ B6 and DiO+ HOD RBCs. Whole blood was collected at multiple time points and (A) the ratio of DiO+ HOD RBCs to DiI+ B6 RBCs was determined to assess allogeneic RBC survival and (B) the overall percentage of DiO+ HOD RBCs was determined. Lines: poly (I:C) are red circles, PBS are blue squares. This experiment was repeated 3 times with 3–5 mice per group with similar results. A representative experiment is shown.

    Techniques Used: Mouse Assay

    Poly (I:C) leads to increased RBC consumption and upregulation of MHCII and CD86 expression. Recipient B6 mice were treated with poly (I:C) or control PBS and subsequently transfused with 100 uL of packed, leukoreduced, DiO-labeled HOD RBCs. At multiple time points, spleens were harvested, collagenase digested, and stained with antibodies to delineate APC subsets. (A) Total splenocyte counts were calculated and (B) the percent of DiO+ leukocytes was determined. The MFI of (C) DiO, (D) MHCII, and (E) CD86 was assessed for individual APC subsets. These experiments were repeated 3 times with 3 mice per group with similar results. A representative experiment is shown. PBS treated animals are shown with an open bar and poly (I:C) treatment is shown with a solid black bar. For analysis, T cells, B cells, and RBCs were excluded from total live leukocytes by gating out cells positive for Thy1.2, CD19, NK1.1, CD49b, and TER119. The following phenotypes were used to delineate APC subsets: RPMs: CD11c −/lo CD11b −/lo F4/80 + ; CD8+ DCs: CD11c hi CD11b − CD8 + ; CD11b+ DCs: CD11c hi CD11b + CD8 − ; pDCs: PDCA1 + CD11c int Ly6C hi ; inflammatory monocytes: CD11c −/lo CD11b + Ly6G − Ly6C hi CD115 + ; resident monocytes: CD11c −/lo CD11b + Ly6G − Ly6C lo CD115 − ; neutrophils: CD11c −/lo CD11b + Ly6G + and a high side scatter. For significance, **** p ≤ 0.0001, *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05.
    Figure Legend Snippet: Poly (I:C) leads to increased RBC consumption and upregulation of MHCII and CD86 expression. Recipient B6 mice were treated with poly (I:C) or control PBS and subsequently transfused with 100 uL of packed, leukoreduced, DiO-labeled HOD RBCs. At multiple time points, spleens were harvested, collagenase digested, and stained with antibodies to delineate APC subsets. (A) Total splenocyte counts were calculated and (B) the percent of DiO+ leukocytes was determined. The MFI of (C) DiO, (D) MHCII, and (E) CD86 was assessed for individual APC subsets. These experiments were repeated 3 times with 3 mice per group with similar results. A representative experiment is shown. PBS treated animals are shown with an open bar and poly (I:C) treatment is shown with a solid black bar. For analysis, T cells, B cells, and RBCs were excluded from total live leukocytes by gating out cells positive for Thy1.2, CD19, NK1.1, CD49b, and TER119. The following phenotypes were used to delineate APC subsets: RPMs: CD11c −/lo CD11b −/lo F4/80 + ; CD8+ DCs: CD11c hi CD11b − CD8 + ; CD11b+ DCs: CD11c hi CD11b + CD8 − ; pDCs: PDCA1 + CD11c int Ly6C hi ; inflammatory monocytes: CD11c −/lo CD11b + Ly6G − Ly6C hi CD115 + ; resident monocytes: CD11c −/lo CD11b + Ly6G − Ly6C lo CD115 − ; neutrophils: CD11c −/lo CD11b + Ly6G + and a high side scatter. For significance, **** p ≤ 0.0001, *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05.

    Techniques Used: Expressing, Mouse Assay, Labeling, Staining

    CD8+ and CD11b+ DCs promote proliferation and immune synapses with HOD RBC-specific T cells. Recipient B6 mice were treated with 200 ug of poly (I:C) or control PBS and subsequently transfused with 100 uL of leukoreduced, packed, DiO-labeled HOD RBCs. Spleens were harvested 18–24 h post transfusion, collagenase digested, and stained with antibodies to delineate individual APC populations. DiO+ APCs were sorted and co-cultured at a 10:1 ratio with CD4 enriched OTII T cells labeled with CFSE-FR. (A) Co-cultured cells were harvested after 3 days and CFSE-FR dilution was assessed in Va2+Vb5.1/5.2+CD4+Thy1.1+ OTII T cells. Lines: pDCs (green), CD11b+ DCs (red), CD8+ DCs (blue), RPM (purple), PMA/ionomycin (black), and media alone (gray). In separate experiments, co-cultured cells were harvested after 2 days and stained with antibodies to identify APCs, OTII T cells, and the immunological synapse. APCs were identified as MHCII+ (I-A b +) and either CD11c+ (for DCs) or F4/80+ (for RPMs) whereas OTIIs were defined as Va2+CD4+Thy1.1+. The immunological synapse was determined by co-expression of Va2, CD4, MHCII, and CD18 (also known as LFA-1). (B) The number and frequency of immunological synapses was determined and (C) representative images shown. These experiments were repeated 3 times with similar results; a representative experiment is shown.
    Figure Legend Snippet: CD8+ and CD11b+ DCs promote proliferation and immune synapses with HOD RBC-specific T cells. Recipient B6 mice were treated with 200 ug of poly (I:C) or control PBS and subsequently transfused with 100 uL of leukoreduced, packed, DiO-labeled HOD RBCs. Spleens were harvested 18–24 h post transfusion, collagenase digested, and stained with antibodies to delineate individual APC populations. DiO+ APCs were sorted and co-cultured at a 10:1 ratio with CD4 enriched OTII T cells labeled with CFSE-FR. (A) Co-cultured cells were harvested after 3 days and CFSE-FR dilution was assessed in Va2+Vb5.1/5.2+CD4+Thy1.1+ OTII T cells. Lines: pDCs (green), CD11b+ DCs (red), CD8+ DCs (blue), RPM (purple), PMA/ionomycin (black), and media alone (gray). In separate experiments, co-cultured cells were harvested after 2 days and stained with antibodies to identify APCs, OTII T cells, and the immunological synapse. APCs were identified as MHCII+ (I-A b +) and either CD11c+ (for DCs) or F4/80+ (for RPMs) whereas OTIIs were defined as Va2+CD4+Thy1.1+. The immunological synapse was determined by co-expression of Va2, CD4, MHCII, and CD18 (also known as LFA-1). (B) The number and frequency of immunological synapses was determined and (C) representative images shown. These experiments were repeated 3 times with similar results; a representative experiment is shown.

    Techniques Used: Mouse Assay, Labeling, Staining, Cell Culture, Expressing

    28) Product Images from "Imaging of human glioblastoma cells and their interactions with mesenchymal stem cells in the zebrafish (Danio rerio) embryonic brain"

    Article Title: Imaging of human glioblastoma cells and their interactions with mesenchymal stem cells in the zebrafish (Danio rerio) embryonic brain

    Journal: Radiology and Oncology

    doi: 10.1515/raon-2016-0017

    Imaging of co-cultures of GBM cells and MSCs in the brain of zebrafish embryos. A mixture of fluorescent-protein-expressing GBM cells and carbocyaninedye-labeled MSCs was implanted into the brain of the zebrafish embryos. Three days after implantation, the embryos were fixed, cleared in Sca l eU2 without the addition of Triton X-100, and imaged with confocal microscopy. (A) The head of a zebrafish embryo with a co-culture of U87-DsRed cells (red) and DiO-labeled MSCs (green) implanted in the brain. (B) The head of a zebrafish embryo with a co-culture of U373-GFP cells (green) and DiI-labeled MSCs (red) implanted in the brain. (C) Invasion of DiI-labeled MSCs (red) along the central canal of the spinal cord. (D) Three-dimensional rendering of a mixed mass of U373 cells (green) and MSCs (red) in a brain obtained from a cleared embryo. Nuclei are stained with methyl green (presented in blue). Scale bars: 250 μm (A, B); 100 μm (C) ; 50 μm (D) .
    Figure Legend Snippet: Imaging of co-cultures of GBM cells and MSCs in the brain of zebrafish embryos. A mixture of fluorescent-protein-expressing GBM cells and carbocyaninedye-labeled MSCs was implanted into the brain of the zebrafish embryos. Three days after implantation, the embryos were fixed, cleared in Sca l eU2 without the addition of Triton X-100, and imaged with confocal microscopy. (A) The head of a zebrafish embryo with a co-culture of U87-DsRed cells (red) and DiO-labeled MSCs (green) implanted in the brain. (B) The head of a zebrafish embryo with a co-culture of U373-GFP cells (green) and DiI-labeled MSCs (red) implanted in the brain. (C) Invasion of DiI-labeled MSCs (red) along the central canal of the spinal cord. (D) Three-dimensional rendering of a mixed mass of U373 cells (green) and MSCs (red) in a brain obtained from a cleared embryo. Nuclei are stained with methyl green (presented in blue). Scale bars: 250 μm (A, B); 100 μm (C) ; 50 μm (D) .

    Techniques Used: Imaging, Expressing, Labeling, Confocal Microscopy, Co-Culture Assay, Staining

    29) Product Images from "Interpreting ciliopathy-associated missense variants of uncertain significance (VUS) in Caenorhabditis elegans"

    Article Title: Interpreting ciliopathy-associated missense variants of uncertain significance (VUS) in Caenorhabditis elegans

    Journal: Human Molecular Genetics

    doi: 10.1093/hmg/ddab344

    Quantitative phenotyping of cilia-dependent phenotypes in C. elegans. All assays were performed blind with at least three independent biological replicates. ( A ) Lipophilic dye (DiI or DiO) filling assay of the four phasmid (tail) neurons. The number of cell bodies which uptake dye was counted (values range from 0 to 4). The bar graph indicates the proportion of the population with dye uptake in 0 (white) to 4 (black) phasmid neurons. The number of worms is shown in brackets. Statistical significance according to Kruskal–Wallis followed by Schaich–Hammerle post hoc test. ( B ) Assessment of worm roaming behaviour normalized to wild-type. A single young adult hermaphrodite was placed on a food-rich plate for 20 h and the roaming activity was quantified. The number of worms is shown in brackets. Box plots indicate the maximum and minimum values (bars), median, lower quartile and upper quartile. Statistical significance according to Kruskal–Wallis followed by Dunn’s post hoc test. ( C ) Quantification of worm chemotaxis towards benzaldehyde after 60 min. Assay is performed on a population of 50–300 worms. The number of assays is shown in brackets. Statistical significance according to ANOVA followed by Tukey’s post hoc test. Box plots indicate the maximum and minimum values (bars), median, lower quartile and upper quartile. * and * * * refer to P -values of
    Figure Legend Snippet: Quantitative phenotyping of cilia-dependent phenotypes in C. elegans. All assays were performed blind with at least three independent biological replicates. ( A ) Lipophilic dye (DiI or DiO) filling assay of the four phasmid (tail) neurons. The number of cell bodies which uptake dye was counted (values range from 0 to 4). The bar graph indicates the proportion of the population with dye uptake in 0 (white) to 4 (black) phasmid neurons. The number of worms is shown in brackets. Statistical significance according to Kruskal–Wallis followed by Schaich–Hammerle post hoc test. ( B ) Assessment of worm roaming behaviour normalized to wild-type. A single young adult hermaphrodite was placed on a food-rich plate for 20 h and the roaming activity was quantified. The number of worms is shown in brackets. Box plots indicate the maximum and minimum values (bars), median, lower quartile and upper quartile. Statistical significance according to Kruskal–Wallis followed by Dunn’s post hoc test. ( C ) Quantification of worm chemotaxis towards benzaldehyde after 60 min. Assay is performed on a population of 50–300 worms. The number of assays is shown in brackets. Statistical significance according to ANOVA followed by Tukey’s post hoc test. Box plots indicate the maximum and minimum values (bars), median, lower quartile and upper quartile. * and * * * refer to P -values of

    Techniques Used: Activity Assay, Chemotaxis Assay

    30) Product Images from "Escherichia coli outer membrane vesicles can contribute to sepsis induced cardiac dysfunction"

    Article Title: Escherichia coli outer membrane vesicles can contribute to sepsis induced cardiac dysfunction

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-16363-9

    OMVs in HL-1 induce cytotoxicity and pro-inflammatory cytokine production. ( A ) OMVs (10 μg/mL) were incubated with cells for 6 h. OMVs, cell membrane, and nuclei were stained by DiO (Green), Cellmask Deep Red (Red), and DAPI, respectively. Scale bars, 20 µm. ( B ) Cells were exposed to LPS (0.6 µg/mL) or OMVs (1, 10 µg/mL) for 48 h and cell viability was assessed by MTT assay. Results are expressed in percentage of control. ( C ) Cell were treated with the peroxide-sensitive florescent probe 2′,7′-dichlorofluorescein di-acetate, followed by incubation with LPS (0.6 µg/mL) or OMVs (1, 10 µg/mL) for 3 h. Fluorescence was read at 560 nm, and ROS generation was expressed as percent change compared to the untreated control. ( D and E ) LPS (0.6 µg/mL) or OMVs (1, 10 µg/mL) were added to the cells, and the culture supernatant concentrations of TNF-α and IL-6 24 h later are shown in panel D and E , respectively. * P
    Figure Legend Snippet: OMVs in HL-1 induce cytotoxicity and pro-inflammatory cytokine production. ( A ) OMVs (10 μg/mL) were incubated with cells for 6 h. OMVs, cell membrane, and nuclei were stained by DiO (Green), Cellmask Deep Red (Red), and DAPI, respectively. Scale bars, 20 µm. ( B ) Cells were exposed to LPS (0.6 µg/mL) or OMVs (1, 10 µg/mL) for 48 h and cell viability was assessed by MTT assay. Results are expressed in percentage of control. ( C ) Cell were treated with the peroxide-sensitive florescent probe 2′,7′-dichlorofluorescein di-acetate, followed by incubation with LPS (0.6 µg/mL) or OMVs (1, 10 µg/mL) for 3 h. Fluorescence was read at 560 nm, and ROS generation was expressed as percent change compared to the untreated control. ( D and E ) LPS (0.6 µg/mL) or OMVs (1, 10 µg/mL) were added to the cells, and the culture supernatant concentrations of TNF-α and IL-6 24 h later are shown in panel D and E , respectively. * P

    Techniques Used: Incubation, Staining, MTT Assay, Fluorescence

    31) Product Images from "Genetic interplay between the transcription factors Sp8 and Emx2 in the patterning of the forebrain"

    Article Title: Genetic interplay between the transcription factors Sp8 and Emx2 in the patterning of the forebrain

    Journal: Neural Development

    doi: 10.1186/1749-8104-2-8

    Caudalized gene expression and thalamic innervation in the brain of Sp8 mutants. (a, a', b, b') WMISH on dissected E12.5 forebrains, using Emx2 and Pax6 riboprobes. The Emx2 gradient is up-regulated in cKO (arrows in (a, a')). The expression level of Pax6 is diminished (arrowheads in (b, b')) in Sp8 mutants. Analysis of the area specific marker genes (c, c') EphrinA5 , (d, d') EphA7 , (e, e') Coup-TF1 and (f, f') ID-2 on E18.5 sagittal sections using ISH (rostral is to the left). The visual cortex area in cKO cortices appears expanded towards the rostral brain, as demonstrated by EphA7 , ID-2 and Coup-TF1 expression (strong rostral domain of EphA7 in (d'), strong ID-2 domain in (f'), strong domain of Coup-TF1 in (e')). The somatosensory cortex (area between the red arrows in (c, d') and between the black and red arrows in (f)) shifts rostrally in Sp8 conditional mutants (compare (c, d) with (c', d'), and (f) with the area indicated by the asterisk in (f')). The motor cortex expression area appears condensed (compare rostral to left arrow in (c, c', d, d')) in Sp8 deficient specimens. (g, g') Coronal sections of E18.5 brains labeled with DiI and DiO and counterstained with DAPI. In controls, DiI, placed in the visual cortex (inset in (g)), retrogradelly labels only cells in the dLGN (g). Placing DiO in the somatosensory cortex (inset in (g)) marks only cells in the VP (g). In the mutants the green dye, placed into somatosensory cortex (inset in (g')) labels cells in the VP and the dLGN (g'). (h) GST-pull down reveals that Emx2 lacking the homeobox (Emx2ΔHox) does not bind GST, GST-Sp8 (GST-Sp8FL) or GST-Sp8 lacking zinc fingers (GST-Sp8ΔZn) (lanes 2–4). Full-length Emx2 protein (Emx2FL) does not bind GST, but interacts with GST-Sp8 (GST-Sp8FL) and GST-SP8 lacking zinc fingers (GST-Sp8ΔZn) (lanes 6–8). Lanes 1 and 5 show 10% of the radiolabeled Emx2 isoforms, used as input for the binding assays in lanes 2–4 and 6–8.
    Figure Legend Snippet: Caudalized gene expression and thalamic innervation in the brain of Sp8 mutants. (a, a', b, b') WMISH on dissected E12.5 forebrains, using Emx2 and Pax6 riboprobes. The Emx2 gradient is up-regulated in cKO (arrows in (a, a')). The expression level of Pax6 is diminished (arrowheads in (b, b')) in Sp8 mutants. Analysis of the area specific marker genes (c, c') EphrinA5 , (d, d') EphA7 , (e, e') Coup-TF1 and (f, f') ID-2 on E18.5 sagittal sections using ISH (rostral is to the left). The visual cortex area in cKO cortices appears expanded towards the rostral brain, as demonstrated by EphA7 , ID-2 and Coup-TF1 expression (strong rostral domain of EphA7 in (d'), strong ID-2 domain in (f'), strong domain of Coup-TF1 in (e')). The somatosensory cortex (area between the red arrows in (c, d') and between the black and red arrows in (f)) shifts rostrally in Sp8 conditional mutants (compare (c, d) with (c', d'), and (f) with the area indicated by the asterisk in (f')). The motor cortex expression area appears condensed (compare rostral to left arrow in (c, c', d, d')) in Sp8 deficient specimens. (g, g') Coronal sections of E18.5 brains labeled with DiI and DiO and counterstained with DAPI. In controls, DiI, placed in the visual cortex (inset in (g)), retrogradelly labels only cells in the dLGN (g). Placing DiO in the somatosensory cortex (inset in (g)) marks only cells in the VP (g). In the mutants the green dye, placed into somatosensory cortex (inset in (g')) labels cells in the VP and the dLGN (g'). (h) GST-pull down reveals that Emx2 lacking the homeobox (Emx2ΔHox) does not bind GST, GST-Sp8 (GST-Sp8FL) or GST-Sp8 lacking zinc fingers (GST-Sp8ΔZn) (lanes 2–4). Full-length Emx2 protein (Emx2FL) does not bind GST, but interacts with GST-Sp8 (GST-Sp8FL) and GST-SP8 lacking zinc fingers (GST-Sp8ΔZn) (lanes 6–8). Lanes 1 and 5 show 10% of the radiolabeled Emx2 isoforms, used as input for the binding assays in lanes 2–4 and 6–8.

    Techniques Used: Expressing, Marker, In Situ Hybridization, Labeling, Zinc-Fingers, Binding Assay

    32) Product Images from "Capture of endothelial cells under flow using immobilized vascular endothelial growth factor"

    Article Title: Capture of endothelial cells under flow using immobilized vascular endothelial growth factor

    Journal: Biomaterials

    doi: 10.1016/j.biomaterials.2015.02.025

    HUVEC capture from complex cell mixtures (A) Capture of EC at low shear (0.5 dyne/cm 2 ) from dual cell mixtures of HUVECs and NIH-3T3 cells of 1:0, 0:1, 1:1, 1:10, or 1:100 ratio. HUVECs and NIH-3T3 were pre-labeled with DIL and DIO, respectively prior to running through the fluidic device. The solid line indicates captured HUVECs and the dotted line captured NIH-3T3 cells. (B) EC capture from a HUVEC:NIH-3T3 mixture (ratio 1:100) under intermediate (5 dyne/cm 2 ) or high shear stress (10 dyne/cm 2 ). (C) HUVEC were pre-labeled with DII and spiked into whole human blood at 5×10 3 cells per mL of blood (HUVEC: Blood cell ratio=10 6 ). Then 1mL of HUVEC containing blood was passed through the micro-channel at high shear stress (10 dyne/cm 2 ). (*) denotes statistical significance (p
    Figure Legend Snippet: HUVEC capture from complex cell mixtures (A) Capture of EC at low shear (0.5 dyne/cm 2 ) from dual cell mixtures of HUVECs and NIH-3T3 cells of 1:0, 0:1, 1:1, 1:10, or 1:100 ratio. HUVECs and NIH-3T3 were pre-labeled with DIL and DIO, respectively prior to running through the fluidic device. The solid line indicates captured HUVECs and the dotted line captured NIH-3T3 cells. (B) EC capture from a HUVEC:NIH-3T3 mixture (ratio 1:100) under intermediate (5 dyne/cm 2 ) or high shear stress (10 dyne/cm 2 ). (C) HUVEC were pre-labeled with DII and spiked into whole human blood at 5×10 3 cells per mL of blood (HUVEC: Blood cell ratio=10 6 ). Then 1mL of HUVEC containing blood was passed through the micro-channel at high shear stress (10 dyne/cm 2 ). (*) denotes statistical significance (p

    Techniques Used: Labeling

    33) Product Images from "Fgf10+ progenitors give rise to the chick hypothalamus by rostral and caudal growth and differentiation"

    Article Title: Fgf10+ progenitors give rise to the chick hypothalamus by rostral and caudal growth and differentiation

    Journal: Development (Cambridge, England)

    doi: 10.1242/dev.153379

    Fate-mapping defines the extent of hypothalamic progenitors. (A-D) The position of the hypothalamus relative to adjacent structures, including Rathke's pouch (RP), shown: (A) in E5 whole-mount side view (dashed lines outline the neuroepithelium and RP); (B) after hemi-dissection at E3; (C) after sagittal sectioning (a high-power view of E5 chick to show the proximity of the infundibulum and RP); and (D) schematically (E5 chick). (E-L) Sagittal sections at E5 (E-H) or E3 (I-L) analysed by double fluorescent in situ hybridisation for Six3 / Foxg1 (E,I) or by single in situ hybridisation for Fgf10 (F,J) or Emx2 (G,K), and illustrated schematically in H,L. Dashed lines in J,K outline RP. Green arrowheads in E,I indicate the telencephalic-hypothalamic boundary. (M-T) Fate-mapping of the medial prosencephalon (M-P) or prosencephalic neck (Q-T). (M,Q) Schematic representation of prosencephalon. White areas indicate the suggested position of hypothalamic progenitors from Garcia-Lopez et al. (2004) (M) or Manning et al. (2006) and Pearson et al. (2011) (Q). Circles show the position of focal DiI (pink) and DiO (green) injections. (N) Dorsal view of a 10-somite embryo after triple injection of DiI in the ventral midline of the medial prosencephalon and of DiO in adjacent basal plate ( n =2). (O,P) Hemi-dissected side view of the same embryo incubated to HH20 (O), schematically represented in P. Labelled cells lie posterior to the optic stalk. (R) Dorsal view of a 9-somite embryo after triple injection of DiI in the ventral midline of the prosencephalic neck and of DiO in adjacent basal plate ( n =2). (S,T) Hemi-dissected side view of the same embryo incubated to HH20 (S), schematically represented in T. DiI-labelled cells populate the ventral hypothalamus, extending from the mammillary pouch to the tuberal hypothalamus. DiO-labelled cells populate the basal plate of p2. Scale bars: 1 mm in A; 200 µm in B; 100 µm in C-S. ant, anterior hypothalamus; cf, cephalic flexure; hyp, hypothalamus; mam, mammillary hypothalamus; mes, mesencephalon; mp, mammillary pouch; os, optic stalk; p1-3, prosomeres 1-3; RP, Rathke's pouch; tel, telencephalon; tub, tuberal hypothalamus.
    Figure Legend Snippet: Fate-mapping defines the extent of hypothalamic progenitors. (A-D) The position of the hypothalamus relative to adjacent structures, including Rathke's pouch (RP), shown: (A) in E5 whole-mount side view (dashed lines outline the neuroepithelium and RP); (B) after hemi-dissection at E3; (C) after sagittal sectioning (a high-power view of E5 chick to show the proximity of the infundibulum and RP); and (D) schematically (E5 chick). (E-L) Sagittal sections at E5 (E-H) or E3 (I-L) analysed by double fluorescent in situ hybridisation for Six3 / Foxg1 (E,I) or by single in situ hybridisation for Fgf10 (F,J) or Emx2 (G,K), and illustrated schematically in H,L. Dashed lines in J,K outline RP. Green arrowheads in E,I indicate the telencephalic-hypothalamic boundary. (M-T) Fate-mapping of the medial prosencephalon (M-P) or prosencephalic neck (Q-T). (M,Q) Schematic representation of prosencephalon. White areas indicate the suggested position of hypothalamic progenitors from Garcia-Lopez et al. (2004) (M) or Manning et al. (2006) and Pearson et al. (2011) (Q). Circles show the position of focal DiI (pink) and DiO (green) injections. (N) Dorsal view of a 10-somite embryo after triple injection of DiI in the ventral midline of the medial prosencephalon and of DiO in adjacent basal plate ( n =2). (O,P) Hemi-dissected side view of the same embryo incubated to HH20 (O), schematically represented in P. Labelled cells lie posterior to the optic stalk. (R) Dorsal view of a 9-somite embryo after triple injection of DiI in the ventral midline of the prosencephalic neck and of DiO in adjacent basal plate ( n =2). (S,T) Hemi-dissected side view of the same embryo incubated to HH20 (S), schematically represented in T. DiI-labelled cells populate the ventral hypothalamus, extending from the mammillary pouch to the tuberal hypothalamus. DiO-labelled cells populate the basal plate of p2. Scale bars: 1 mm in A; 200 µm in B; 100 µm in C-S. ant, anterior hypothalamus; cf, cephalic flexure; hyp, hypothalamus; mam, mammillary hypothalamus; mes, mesencephalon; mp, mammillary pouch; os, optic stalk; p1-3, prosomeres 1-3; RP, Rathke's pouch; tel, telencephalon; tub, tuberal hypothalamus.

    Techniques Used: Dissection, In Situ, Hybridization, Injection, Incubation

    34) Product Images from "Reconstruction of Auto-Tissue-Engineered Lamellar Cornea by Dynamic Culture for Transplantation: A Rabbit Model"

    Article Title: Reconstruction of Auto-Tissue-Engineered Lamellar Cornea by Dynamic Culture for Transplantation: A Rabbit Model

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0093012

    Physiological function of ATELC-Dynamic in a rabbit lamellar keratoplasty model. (A) Postoperative observation of lamellar keratoplasty. (B) The light transmittance of transplanted lamellar cornea over the wavelength range of 300–800 nm at 20 days. (C) DiO-labeled seeding cells at 7 days, and the expression of collagen III at 20 days.
    Figure Legend Snippet: Physiological function of ATELC-Dynamic in a rabbit lamellar keratoplasty model. (A) Postoperative observation of lamellar keratoplasty. (B) The light transmittance of transplanted lamellar cornea over the wavelength range of 300–800 nm at 20 days. (C) DiO-labeled seeding cells at 7 days, and the expression of collagen III at 20 days.

    Techniques Used: Labeling, Expressing

    35) Product Images from "Cadherin-mediated cell sorting not determined by binding or adhesion specificity"

    Article Title: Cadherin-mediated cell sorting not determined by binding or adhesion specificity

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200108040

    Variable sorting specificities for different cadherin-expressing CHO cells. Cell aggregation assays using cells labeled with fluorescent dyes, either diI (red) or diO (green). Cells were allowed to aggregate for 3 h. For A–C, examples of fluorescence are shown in top panels, with quantification of sorting versus mixing shown in the graphs below. (A) diI-labeled C-CHO cells mix completely with diO-labeled C-CHO cells, and diI-labeled HE-CHO cells also mix completely with diO-labeled HE-CHO cells, showing that the fluorescent label does not cause cells to sort artifactually. Middle, C-CHO cells (red) also mix to a large extent with HE-CHO cells (green). (B) XE-CHO cells (red in all cases) mix with XE-CHO cells (green) or with C-CHO cells (green), but sort out from HE-CHO cells (green). (C) HN-CHO cells (red in all cases) mix with HN-CHO cells (green) or C-CHO cells (green), but sort out from HE-CHO cells (green). Total number of counted aggregates is set at 100%. Black bars, diI labeled aggregates (red); striped bars, diI- and diO-labeled mixed aggregates; white bars, diO-labeled aggregates (green).
    Figure Legend Snippet: Variable sorting specificities for different cadherin-expressing CHO cells. Cell aggregation assays using cells labeled with fluorescent dyes, either diI (red) or diO (green). Cells were allowed to aggregate for 3 h. For A–C, examples of fluorescence are shown in top panels, with quantification of sorting versus mixing shown in the graphs below. (A) diI-labeled C-CHO cells mix completely with diO-labeled C-CHO cells, and diI-labeled HE-CHO cells also mix completely with diO-labeled HE-CHO cells, showing that the fluorescent label does not cause cells to sort artifactually. Middle, C-CHO cells (red) also mix to a large extent with HE-CHO cells (green). (B) XE-CHO cells (red in all cases) mix with XE-CHO cells (green) or with C-CHO cells (green), but sort out from HE-CHO cells (green). (C) HN-CHO cells (red in all cases) mix with HN-CHO cells (green) or C-CHO cells (green), but sort out from HE-CHO cells (green). Total number of counted aggregates is set at 100%. Black bars, diI labeled aggregates (red); striped bars, diI- and diO-labeled mixed aggregates; white bars, diO-labeled aggregates (green).

    Techniques Used: Expressing, Labeling, Fluorescence

    36) Product Images from "A co-culture system of rat synovial stem cells and meniscus cells promotes cell proliferation and differentiation as compared to mono-culture"

    Article Title: A co-culture system of rat synovial stem cells and meniscus cells promotes cell proliferation and differentiation as compared to mono-culture

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-25709-w

    Cell morphology and cell growth in co-culture systems were examined by confocal laser scanning microscopy. MCs and SMSCs were stained with DiI and DiO, respectively, to display two colours (DiI: SMSCs, DiO: MCs). After co-culturing for 7 days, the two types of cells at different ratios reached confluence ( B – D ). In the co-culture groups, for example, a ratio of 3:1 means that the number of SMSCs was 3 times that of MCs. SMSCs had a spindle shape in the co-culture groups (red), while MCs had a polygonal shape both in the mono-culture control group and in the co-culture groups (green). Magnification: ×100.
    Figure Legend Snippet: Cell morphology and cell growth in co-culture systems were examined by confocal laser scanning microscopy. MCs and SMSCs were stained with DiI and DiO, respectively, to display two colours (DiI: SMSCs, DiO: MCs). After co-culturing for 7 days, the two types of cells at different ratios reached confluence ( B – D ). In the co-culture groups, for example, a ratio of 3:1 means that the number of SMSCs was 3 times that of MCs. SMSCs had a spindle shape in the co-culture groups (red), while MCs had a polygonal shape both in the mono-culture control group and in the co-culture groups (green). Magnification: ×100.

    Techniques Used: Co-Culture Assay, Confocal Laser Scanning Microscopy, Staining

    37) Product Images from "Dinaciclib induces immunogenic cell death and enhances anti-PD1–mediated tumor suppression"

    Article Title: Dinaciclib induces immunogenic cell death and enhances anti-PD1–mediated tumor suppression

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI94586

    Dinaciclib-treated tumor cells enhance DC function. DiO-labeled CT26 cells were treated with the indicated concentrations of dinaciclib for 24 hours and then cocultured with BMDCs for an additional 24 hours. ( A ) The percentage of CD11c + DCs with engulfed tumor cells was assessed by flow cytometry, as was the expression of ( B ) MHCII, ( C ) CD86, and ( D ) CD80 on CD11c + DCs after coculture. ( E ) Secretion of IL-1β into the coculture supernatant was determined by MSD assay. Data represent the mean value ± SEM of 3 to 4 replicates from 1 representative experiment. *** P
    Figure Legend Snippet: Dinaciclib-treated tumor cells enhance DC function. DiO-labeled CT26 cells were treated with the indicated concentrations of dinaciclib for 24 hours and then cocultured with BMDCs for an additional 24 hours. ( A ) The percentage of CD11c + DCs with engulfed tumor cells was assessed by flow cytometry, as was the expression of ( B ) MHCII, ( C ) CD86, and ( D ) CD80 on CD11c + DCs after coculture. ( E ) Secretion of IL-1β into the coculture supernatant was determined by MSD assay. Data represent the mean value ± SEM of 3 to 4 replicates from 1 representative experiment. *** P

    Techniques Used: Labeling, Flow Cytometry, Cytometry, Expressing

    38) Product Images from "Therapeutic microparticles functionalized with biomimetic cardiac stem cell membranes and secretome"

    Article Title: Therapeutic microparticles functionalized with biomimetic cardiac stem cell membranes and secretome

    Journal: Nature Communications

    doi: 10.1038/ncomms13724

    Physiochemical and biological properties of CMMPs. ( a ) Overall biochemical design and study model of CMMPs. MPs (that is, Control MP 1 ) were fabricated from PLGA and conditioned media of human CSCs, then MPs were cloaked with membrane fragments of CSCs to form CMMPs. Control MP 2 was fabricated by cloaking empty PLGA particles with CSC membranes. The therapeutic potential of CMMPs was tested in a mouse model of myocardial infarction. ( b , c ) Texas red succinimidyl ester-labelled MPs ( b , red) were cloaked with the membrane fragments of green fluorescent DiO-labelled CSCs ( b , green) to form CMMP ( c , red particle with green coat). Scale bar, 20 μm. ( d , e ) SEM revealed the CSC membrane fragments on CMMPs ( e ) but not on Control MP 1 (non-cloaked MP) ( d ). Scale bar, 10 μm. ( f , g ) Major human CSC markers CD105 ( f ) and CD90 ( g ) were positive on CMMPs and Control MP 2 but not on non-cloaked Control MP 1 , indicating the successful membrane cloaking on CMMPs. ( h ) CMMPs, Control MP 1 and Control MP 2 have similar sizes to those of CSCs. n =3 for each group. ( i ) CMMPs and Control MP 2 carried similar surface antigens as CSCs did. n =3 for each group. ( j – l ) Similar release profile of CSC factors (namely vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF)-1 and hepatocyte growth factor (HGF)) was observed in CMMPs and Control MP 1 , indicating membrane cloaking did not affect the release of CSC factors from CMMPs and Control MP 1 . n =3 for each time point. All data are mean±s.d. Comparisons between any two groups were performed using two-tailed unpaired Student’s t -test. Comparisons among more than two groups were performed using one-way ANOVA followed by post hoc Bonferroni test.
    Figure Legend Snippet: Physiochemical and biological properties of CMMPs. ( a ) Overall biochemical design and study model of CMMPs. MPs (that is, Control MP 1 ) were fabricated from PLGA and conditioned media of human CSCs, then MPs were cloaked with membrane fragments of CSCs to form CMMPs. Control MP 2 was fabricated by cloaking empty PLGA particles with CSC membranes. The therapeutic potential of CMMPs was tested in a mouse model of myocardial infarction. ( b , c ) Texas red succinimidyl ester-labelled MPs ( b , red) were cloaked with the membrane fragments of green fluorescent DiO-labelled CSCs ( b , green) to form CMMP ( c , red particle with green coat). Scale bar, 20 μm. ( d , e ) SEM revealed the CSC membrane fragments on CMMPs ( e ) but not on Control MP 1 (non-cloaked MP) ( d ). Scale bar, 10 μm. ( f , g ) Major human CSC markers CD105 ( f ) and CD90 ( g ) were positive on CMMPs and Control MP 2 but not on non-cloaked Control MP 1 , indicating the successful membrane cloaking on CMMPs. ( h ) CMMPs, Control MP 1 and Control MP 2 have similar sizes to those of CSCs. n =3 for each group. ( i ) CMMPs and Control MP 2 carried similar surface antigens as CSCs did. n =3 for each group. ( j – l ) Similar release profile of CSC factors (namely vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF)-1 and hepatocyte growth factor (HGF)) was observed in CMMPs and Control MP 1 , indicating membrane cloaking did not affect the release of CSC factors from CMMPs and Control MP 1 . n =3 for each time point. All data are mean±s.d. Comparisons between any two groups were performed using two-tailed unpaired Student’s t -test. Comparisons among more than two groups were performed using one-way ANOVA followed by post hoc Bonferroni test.

    Techniques Used: Two Tailed Test

    39) Product Images from "Structural Characterization of Clostridium sordellii Spores of Diverse Human, Animal, and Environmental Origin and Comparison to Clostridium difficile Spores"

    Article Title: Structural Characterization of Clostridium sordellii Spores of Diverse Human, Animal, and Environmental Origin and Comparison to Clostridium difficile Spores

    Journal: mSphere

    doi: 10.1128/mSphere.00343-17

    Spores of strain ATCC 9714 imaged by SEM (A), cryo-EM (B), and 3D-SIM (C and D) with spores labeled with lipid dyes DiO and FM 4-64FX (C) or DiO and spore-specific antibodies (D). The 3D-SIM image presented in panel D is a maximum-intensity projection of the z series through the spore volume (main image), supplemented by the xz projection (bottom panel) and yz projection (right panel). The red cross marks the distal end of one spore pole, the yellow line marks the x plane, and the blue line marks the y plane. The exosporial openings are visible in the xz and yz projections, where the spore has been sliced along the yellow line and is viewed from the top or where the spore has been sliced along the blue line and is viewed from the side of the spore, respectively. IS, inner spore; Ex, exosporium. Bar, 0.5 µm.
    Figure Legend Snippet: Spores of strain ATCC 9714 imaged by SEM (A), cryo-EM (B), and 3D-SIM (C and D) with spores labeled with lipid dyes DiO and FM 4-64FX (C) or DiO and spore-specific antibodies (D). The 3D-SIM image presented in panel D is a maximum-intensity projection of the z series through the spore volume (main image), supplemented by the xz projection (bottom panel) and yz projection (right panel). The red cross marks the distal end of one spore pole, the yellow line marks the x plane, and the blue line marks the y plane. The exosporial openings are visible in the xz and yz projections, where the spore has been sliced along the yellow line and is viewed from the top or where the spore has been sliced along the blue line and is viewed from the side of the spore, respectively. IS, inner spore; Ex, exosporium. Bar, 0.5 µm.

    Techniques Used: Labeling

    40) Product Images from "“High-Throughput Characterization of Region-Specific Mitochondrial Function and Morphology”"

    Article Title: “High-Throughput Characterization of Region-Specific Mitochondrial Function and Morphology”

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-05152-z

    Characterization of tissue-specific expression and derivation of “regions of interest”. ( A ) Median profiles derived from various C . elegans transgenic lines expressing a fluorescent marker in each respective tissue. Animals were concurrently live-stained with DiO or DiI (which labels sensory neurons) to enable consistent orientation across strains. ( B ) Graphical representation of transgenic lines in ( A ) to demonstrate resolution of individual tissues as a function of the animal’s length. ( C ) Difference (Δ) plot (top) derived from average profiles in ( A ) and significance plot (bottom) based on these differences. For significance plots “−1.3-log 10 ( P-value )” was plotted which distinguished regions of significance by Wilcoxon Rank Sum Test. A value ≥ 0 is equilvalent to P ≤ 0.05. ( D ) Anatomical representation of various C . elegans tissues and the derived “regions of interest” from them (e.g. Anterior: 0–20%, Middle: 20–85%, and Posterior: 85–100%). Pharynx in red, neurons in orange, muscle in green, and intestine in grey. ( E ) Simplified schematic of these three regions, Anterior (white), Middle (grey), and Posterior (dark grey) to demonstrate the relative cutoff points of these various areas of interest. All animals are positioned with the anterior side to the left. Plots are representative of three biological replicates and derived from n ≥ 50 organisms. Sample size ( n ) for all experiments and exact P-values can be found in the Supplementary Table. L.A. Daniele provided the illustrations.
    Figure Legend Snippet: Characterization of tissue-specific expression and derivation of “regions of interest”. ( A ) Median profiles derived from various C . elegans transgenic lines expressing a fluorescent marker in each respective tissue. Animals were concurrently live-stained with DiO or DiI (which labels sensory neurons) to enable consistent orientation across strains. ( B ) Graphical representation of transgenic lines in ( A ) to demonstrate resolution of individual tissues as a function of the animal’s length. ( C ) Difference (Δ) plot (top) derived from average profiles in ( A ) and significance plot (bottom) based on these differences. For significance plots “−1.3-log 10 ( P-value )” was plotted which distinguished regions of significance by Wilcoxon Rank Sum Test. A value ≥ 0 is equilvalent to P ≤ 0.05. ( D ) Anatomical representation of various C . elegans tissues and the derived “regions of interest” from them (e.g. Anterior: 0–20%, Middle: 20–85%, and Posterior: 85–100%). Pharynx in red, neurons in orange, muscle in green, and intestine in grey. ( E ) Simplified schematic of these three regions, Anterior (white), Middle (grey), and Posterior (dark grey) to demonstrate the relative cutoff points of these various areas of interest. All animals are positioned with the anterior side to the left. Plots are representative of three biological replicates and derived from n ≥ 50 organisms. Sample size ( n ) for all experiments and exact P-values can be found in the Supplementary Table. L.A. Daniele provided the illustrations.

    Techniques Used: Expressing, Derivative Assay, Transgenic Assay, Marker, Staining

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    Thermo Fisher dio
    A biosafety assessment of EVs after exposure of kombucha multimicrobial culture (KMC) under space/Mars-like stressors outboard the ISS and <t>OMVs</t> of K. intermedius , isolated from post-flight KMCs. (A) A micrograph of OMVs stained with the lipophilic dye <t>DiO</t> visible in murine fibroblasts. Fibroblast nuclei were stained with DAPI. (1, 2) – OMVs of K. intermedius from initial KMC; (3, 4) – OMVs of K. intermedius from top-located KMC (tKMC). Scale bar, 10 μm. (B) The environment level of ʟ-[ 14 C] glutamate in the preparations of rat brain nerve terminals after co-cultivation with EVs from post-flight KMCs. (C) Survival rate of murine embryo fibroblasts after co-cultivation with different concentrations of OMVs from K. intermedius isolated from tKMC. (D) Survival rate of colorectal carcinoma cells COLO 205 after co-cultivation with different concentrations of OMVs/ K. intermedius , isolated from KMCs exposed outboard the ISS. (E) Endotoxin activity of EVs/KMCs detected with the Limulus amebocyte lysate (LAL) assay compared to standard endotoxin activity from Escherichia coli . (F) Endotoxin activity of OMVs/ K. intermedius detected with the LAL assay. Data were shown as mean ± SD ( n = 3), * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.
    Dio, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher dil vybrant lipophilic cell membrane dyes
    Microdevice operation and modelling of the tumour microenvironment. ( A ) Appearance of the microdevice. Microdevices are fabricated by injection moulding and can be attached to the bottom of a Petri dish using biocompatible adhesive; three identical devices are shown. ( B ) Magnified image of a microdevice with collagen hydrogel confined to the central microchamber and blue-coloured water perfused through the two lateral microchannels to ease visualization. Droplets are located on top of the inlets to prevent evaporation. Scale bar is 1 cm. ( C ) The principle of the live ‘tumour slice’: Culture medium perfused through the lateral microchannels provides nutrients and oxygen creating physiological gradients across the device. Cells near the ‘surrogate’ blood vessels are viable, whereas oxygen-poor cells in the centre of device start to die creating a ‘necrotic core’ similar to the necrotic regions of tumours. ( D ) Cellular visualization in the microdevice. 20 million U-251 MG cells/ml were embedded in collagen and pipetted into the central chamber of the microdevice. Image shows appearance by confocal microscopy 24 h later. Cells were labelled before injection with the green-fluorescent lipid dye Dio <t>Vybrant®</t> which stains cell membranes enabling all cells to be visualized. Scale bar is 400 μm. ( E ) Incomplete cell visualization within a multicellular spheroid due to its thickness. 10000 U-251 MG cells were labelled with green-fluorescent Dio Vybrant® dye, in suspension to ensure all cells were equally labelled and these were used to form the spheroid. Scale bar is 400 μm. ( F ) Quantification of cellular fluorescence across the yellow bordered regions in the microdevice and the spheroid as indicated in ( D,E ).
    Dil Vybrant Lipophilic Cell Membrane Dyes, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    A biosafety assessment of EVs after exposure of kombucha multimicrobial culture (KMC) under space/Mars-like stressors outboard the ISS and OMVs of K. intermedius , isolated from post-flight KMCs. (A) A micrograph of OMVs stained with the lipophilic dye DiO visible in murine fibroblasts. Fibroblast nuclei were stained with DAPI. (1, 2) – OMVs of K. intermedius from initial KMC; (3, 4) – OMVs of K. intermedius from top-located KMC (tKMC). Scale bar, 10 μm. (B) The environment level of ʟ-[ 14 C] glutamate in the preparations of rat brain nerve terminals after co-cultivation with EVs from post-flight KMCs. (C) Survival rate of murine embryo fibroblasts after co-cultivation with different concentrations of OMVs from K. intermedius isolated from tKMC. (D) Survival rate of colorectal carcinoma cells COLO 205 after co-cultivation with different concentrations of OMVs/ K. intermedius , isolated from KMCs exposed outboard the ISS. (E) Endotoxin activity of EVs/KMCs detected with the Limulus amebocyte lysate (LAL) assay compared to standard endotoxin activity from Escherichia coli . (F) Endotoxin activity of OMVs/ K. intermedius detected with the LAL assay. Data were shown as mean ± SD ( n = 3), * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.

    Journal: Frontiers in Microbiology

    Article Title: Fitness of Outer Membrane Vesicles From Komagataeibacter intermedius Is Altered Under the Impact of Simulated Mars-like Stressors Outside the International Space Station

    doi: 10.3389/fmicb.2020.01268

    Figure Lengend Snippet: A biosafety assessment of EVs after exposure of kombucha multimicrobial culture (KMC) under space/Mars-like stressors outboard the ISS and OMVs of K. intermedius , isolated from post-flight KMCs. (A) A micrograph of OMVs stained with the lipophilic dye DiO visible in murine fibroblasts. Fibroblast nuclei were stained with DAPI. (1, 2) – OMVs of K. intermedius from initial KMC; (3, 4) – OMVs of K. intermedius from top-located KMC (tKMC). Scale bar, 10 μm. (B) The environment level of ʟ-[ 14 C] glutamate in the preparations of rat brain nerve terminals after co-cultivation with EVs from post-flight KMCs. (C) Survival rate of murine embryo fibroblasts after co-cultivation with different concentrations of OMVs from K. intermedius isolated from tKMC. (D) Survival rate of colorectal carcinoma cells COLO 205 after co-cultivation with different concentrations of OMVs/ K. intermedius , isolated from KMCs exposed outboard the ISS. (E) Endotoxin activity of EVs/KMCs detected with the Limulus amebocyte lysate (LAL) assay compared to standard endotoxin activity from Escherichia coli . (F) Endotoxin activity of OMVs/ K. intermedius detected with the LAL assay. Data were shown as mean ± SD ( n = 3), * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.

    Article Snippet: Cellular Uptake of Fluorescently Labeled OMVsIsolated OMVs were labeled with DiO (3,3′-dioctadecyloxacarbocyanine perchlorate; Thermo Fisher Scientific, USA), a fluorescent lipophilic stain, with 20 μg/ml.

    Techniques: Isolation, Staining, Activity Assay, LAL Assay

    hucMSC-Ex Reduced Oxidative Stress and Increased Cell Viability in LO2 Hepatocytes after CCl 4 and H 2 O 2 Treatment (A) Location of CM-Dil-labeled exosomes (red) in CM-Dio-labeled L02 cells (green) detected by imaging flow cytometry. L02 cells were CM-Dil/CM-Dio positive (i), and CM-Dil-labeled exosomes were found in CM-Dio-labeled L02 cells (ii). (B) ROS production in CCl 4 - and H 2 O 2 -injured L02 cells detected by DCF probe staining after treatment with PBS, HFL-CM, HFL-Ex, MSC-CM, and huMSC-Ex (MSC-Ex). Representative images of DCF fluorescence in CCl 4 -injured L02 cells (i) and relative DCF fluorescent values showed reduced ROS production in MSC-Ex treated L02 cells (n = 5; ***p

    Journal: Molecular Therapy

    Article Title: hucMSC Exosome-Derived GPX1 Is Required for the Recovery of Hepatic Oxidant Injury

    doi: 10.1016/j.ymthe.2016.11.019

    Figure Lengend Snippet: hucMSC-Ex Reduced Oxidative Stress and Increased Cell Viability in LO2 Hepatocytes after CCl 4 and H 2 O 2 Treatment (A) Location of CM-Dil-labeled exosomes (red) in CM-Dio-labeled L02 cells (green) detected by imaging flow cytometry. L02 cells were CM-Dil/CM-Dio positive (i), and CM-Dil-labeled exosomes were found in CM-Dio-labeled L02 cells (ii). (B) ROS production in CCl 4 - and H 2 O 2 -injured L02 cells detected by DCF probe staining after treatment with PBS, HFL-CM, HFL-Ex, MSC-CM, and huMSC-Ex (MSC-Ex). Representative images of DCF fluorescence in CCl 4 -injured L02 cells (i) and relative DCF fluorescent values showed reduced ROS production in MSC-Ex treated L02 cells (n = 5; ***p

    Article Snippet: Exosomes and L02 cells were labeled with CM-Dil and CM-Dio (Molecular Probes, ThermoFisher) separately according to the manufacturer’s protocol.

    Techniques: Labeling, Imaging, Flow Cytometry, Cytometry, Staining, Fluorescence

    Microdevice operation and modelling of the tumour microenvironment. ( A ) Appearance of the microdevice. Microdevices are fabricated by injection moulding and can be attached to the bottom of a Petri dish using biocompatible adhesive; three identical devices are shown. ( B ) Magnified image of a microdevice with collagen hydrogel confined to the central microchamber and blue-coloured water perfused through the two lateral microchannels to ease visualization. Droplets are located on top of the inlets to prevent evaporation. Scale bar is 1 cm. ( C ) The principle of the live ‘tumour slice’: Culture medium perfused through the lateral microchannels provides nutrients and oxygen creating physiological gradients across the device. Cells near the ‘surrogate’ blood vessels are viable, whereas oxygen-poor cells in the centre of device start to die creating a ‘necrotic core’ similar to the necrotic regions of tumours. ( D ) Cellular visualization in the microdevice. 20 million U-251 MG cells/ml were embedded in collagen and pipetted into the central chamber of the microdevice. Image shows appearance by confocal microscopy 24 h later. Cells were labelled before injection with the green-fluorescent lipid dye Dio Vybrant® which stains cell membranes enabling all cells to be visualized. Scale bar is 400 μm. ( E ) Incomplete cell visualization within a multicellular spheroid due to its thickness. 10000 U-251 MG cells were labelled with green-fluorescent Dio Vybrant® dye, in suspension to ensure all cells were equally labelled and these were used to form the spheroid. Scale bar is 400 μm. ( F ) Quantification of cellular fluorescence across the yellow bordered regions in the microdevice and the spheroid as indicated in ( D,E ).

    Journal: Scientific Reports

    Article Title: Development and characterization of a microfluidic model of the tumour microenvironment

    doi: 10.1038/srep36086

    Figure Lengend Snippet: Microdevice operation and modelling of the tumour microenvironment. ( A ) Appearance of the microdevice. Microdevices are fabricated by injection moulding and can be attached to the bottom of a Petri dish using biocompatible adhesive; three identical devices are shown. ( B ) Magnified image of a microdevice with collagen hydrogel confined to the central microchamber and blue-coloured water perfused through the two lateral microchannels to ease visualization. Droplets are located on top of the inlets to prevent evaporation. Scale bar is 1 cm. ( C ) The principle of the live ‘tumour slice’: Culture medium perfused through the lateral microchannels provides nutrients and oxygen creating physiological gradients across the device. Cells near the ‘surrogate’ blood vessels are viable, whereas oxygen-poor cells in the centre of device start to die creating a ‘necrotic core’ similar to the necrotic regions of tumours. ( D ) Cellular visualization in the microdevice. 20 million U-251 MG cells/ml were embedded in collagen and pipetted into the central chamber of the microdevice. Image shows appearance by confocal microscopy 24 h later. Cells were labelled before injection with the green-fluorescent lipid dye Dio Vybrant® which stains cell membranes enabling all cells to be visualized. Scale bar is 400 μm. ( E ) Incomplete cell visualization within a multicellular spheroid due to its thickness. 10000 U-251 MG cells were labelled with green-fluorescent Dio Vybrant® dye, in suspension to ensure all cells were equally labelled and these were used to form the spheroid. Scale bar is 400 μm. ( F ) Quantification of cellular fluorescence across the yellow bordered regions in the microdevice and the spheroid as indicated in ( D,E ).

    Article Snippet: Fluorescent cell labelling Dio and Dil Vybrant® lipophilic cell membrane dyes (Life technologies, V-22886 and V-22885) were used to fluorescently label cells green or red respectively as per the manufacturer’s instructions.

    Techniques: Injection, Evaporation, Confocal Microscopy, Fluorescence

    Confocal microscopy images of the 3D cultured pHTMC. Analysis of the effect of the ROCK inhibitor Y27632 at 25 nM for 24 h and the effect of LT at 0.5 μg/mL on primary human pHTMC treated with TGF-β2 at 5 ng/mL for 48 h. The cells were treated with TMCM alone (vehicle), 5 ng/mL of TGF-β2 for 48 h, or with TGF-β2 (5 ng/mL) for 24 h followed by a combination of TGF-β2 at 5 ng/mL and with Y-27632 (25 nM) or LT (0.5 µg/mL) for 24 h. Actin fibers are stained in red by phalloidin, membranes with DiO (green), and nuclei with DAPI (blue). Magnification 200×. Scale bar = 30 µm.

    Journal: Journal of Clinical Medicine

    Article Title: The Dual Effect of Rho-Kinase Inhibition on Trabecular Meshwork Cells Cytoskeleton and Extracellular Matrix in an In Vitro Model of Glaucoma

    doi: 10.3390/jcm11041001

    Figure Lengend Snippet: Confocal microscopy images of the 3D cultured pHTMC. Analysis of the effect of the ROCK inhibitor Y27632 at 25 nM for 24 h and the effect of LT at 0.5 μg/mL on primary human pHTMC treated with TGF-β2 at 5 ng/mL for 48 h. The cells were treated with TMCM alone (vehicle), 5 ng/mL of TGF-β2 for 48 h, or with TGF-β2 (5 ng/mL) for 24 h followed by a combination of TGF-β2 at 5 ng/mL and with Y-27632 (25 nM) or LT (0.5 µg/mL) for 24 h. Actin fibers are stained in red by phalloidin, membranes with DiO (green), and nuclei with DAPI (blue). Magnification 200×. Scale bar = 30 µm.

    Article Snippet: The pHTMC stained with DiO (Vybrant™ DiO Cell-Labeling Solution, Invitrogen, V-22886) were gently mixed at a concentration of 105 cells/mL in Matrigel® diluted 1/2 in the TMCM culture medium and then sewn onto inserts in a 12-well plate (Greiner Bio-One ThinCert cell culture insert for 12 well plates, sterile, polyethylene terephthalate (PET) transparent membrane, pore diameter: 0.4 µm.

    Techniques: Confocal Microscopy, Cell Culture, Staining