prosense® 680 probe  (ProSense Inc)

Journal: PLoS ONE

Article Title: Live Imaging of Cysteine-Cathepsin Activity Reveals Dynamics of Focal Inflammation, Angiogenesis, and Polyp Growth

doi: 10.1371/journal.pone.0002916

Figure Lengend Snippet: A small loop of the intestine of each APC Δ468 and wt animals was surgically exposed and underwent laser scanning Intravital Fluorescent Microscopy (IVFM) 24 hour after injection with the ProSense-680. ProSense-680 image of cathepsin activity in (a) healthy intestine, (b) APC Δ468 polyp. AngioSense 750 image of vasculature in (c) healthy intestine, (d) APC Δ468 polyp. Spectrally resolved auto-fluorescence of (e) healthy intestine, (f) APC Δ468 polyp at 505–510 nm. Z-stack slice 42 for the healthy intestine and slice 51 for the APC Δ468 polyp were imaged using an UplanApo 10× objective with 2× electronic zoom (pixel size 1.05 µm with resolution 2.42 µm). (g) The mean volume of particles visualized at 694 nm (ProSense-680); green bar represents APC Δ468 polyps (128,900±104,800 µm 3 (n = 92)), red bar healthy intestine (4,538±797.5 µm 3 (n = 81)). (h) The mean volume of particles visualized at 790 nm (AngioSense-750); green bar represents vasculature in polyps (11270±3207 µm 3 , n = 101), red bar represents vasculature in healthy intestine (4538±798 µm 3 , n = 81). Calculated with the Image J “3D particle analysis” plug-in. (i) Linear regression, correlating the size of ProSense-680 particles in polyps (green dots, 1/slope = 0.004540, r 2 = 1) with those in healthy intestine (red dots, 1/slope = 0.004539, r 2 = 0.9999); note that the increase in intensity corresponds to the increase in the number of cathepsin active cells. (j) “Calculated centers of intensity” of particles in the z-axis of the APC Δ468 adenoma (green diamonds) and healthy intestine (red triangles); note distribution throughout the z- stack, and that the total intensity of the particles in the APC Δ468 adenoma is at least 2 orders of magnitude higher (mean total intensity 1.322×10 7 ±8.881×10 6 units) than the particles in the wt intestine (mean total intensity 8592±1257 units, P <0.0001 one sample t test).

Article Snippet: Both of these myeloid cell types contributed equally to the local activation of the ProSense 680 probe.

Techniques: Microscopy, Injection, Activity Assay, Fluorescence

Journal: PLoS ONE

Article Title: Live Imaging of Cysteine-Cathepsin Activity Reveals Dynamics of Focal Inflammation, Angiogenesis, and Polyp Growth

doi: 10.1371/journal.pone.0002916

Figure Lengend Snippet: Cryosections of ProSense-680 in vivo stained intestine from APC Δ468 mice were stained with antibodies to CD11b (AlexaFluor 488), Gr1 (AlexaFluor 594) and DAPI. The merged images of CD11b with DAPI (a, CD11b green, DAPI gray), Gr1 with DAPI (b, Gr1 red, DAPI gray), and ProSense-680 with DAPI (c, ProSense-680 blue, DAPI gray) were produced with the “RGB gray” plug-in of Image J. The “colocalization finder” plug-in produced images where the colocalized pixels appear white while the ProSense-680 was red (d&e), the CD11b was green (d, colocalization analysis of ProSense-680 and CD11b staining) and the Gr1 was green (e, colocalization analysis of ProSense-680 and Gr1 staining). ×400 magnification. Arrows mark a CD11b + Gr1 ProSense 680 + cell. Representative FACS dot-plots of MNCs prepared from polyposis intestine and ex vivo stained with ProSense-680 followed by CD11b and Gr1 staining. The live MNCs were gated for ProSence-680 + cells, which were analyzed for CD11b + Gr1 + (f, MDSCs) and CD11b + F4/80 + (g, macrophages) cells. Cumulative results of 6 FACS experiments showing % of CD11b + Gr1 + ProSense-680 + and CD11b + F4/80 + ProSense 680 + among total infiltrating MNCs. Note that among the ProSense-680 + cells (11±0.69% of total MNCs) over 75% were either CD11b + Gr1 + (3.4±0.6% of total MNCs) or CD11b + F4/80 (5.0±0.34% of total MNCs).

Article Snippet: Both of these myeloid cell types contributed equally to the local activation of the ProSense 680 probe.

Techniques: In Vivo, Staining, Produced, Ex Vivo

Journal: PLoS ONE

Article Title: Live Imaging of Cysteine-Cathepsin Activity Reveals Dynamics of Focal Inflammation, Angiogenesis, and Polyp Growth

doi: 10.1371/journal.pone.0002916

Figure Lengend Snippet: (a) Non-linear regression analysis of polyp number and diameter, assuming Gaussian distribution; APC Δ468 Ctsb −/− (continued line, open squares), and APC Δ468 (dotted line, closed triangles). Note that Cathepsin B −/− mice had fewer and smaller polyps. (b) Frequencies of ProSense-680 active leukocytes amongst total MNCs prepared from the intestine of APC Δ468 (open bar, 6.7%±086%) or APC Δ468 Ctsb −/− (filled bar, 11%±0.69%, P = 0.0037; unpaired t test with Welsh correction). (c) Frequencies of ProSense-680 active CD11b + Gr1 + (mean 0.56%) or CD11b + F4/80 + (mean 4.46%) cells, from the intestines of APC Δ468 (open bar) and APC Δ468 Ctsb −/− mice (filled bars); P<0.001, n = 6, 2way ANOVA. Note that Cathepsin B deficiency predominantly impacted the abundance of CD11b + Gr1 + cells. (d) Attenuation of polyposis in anti-TNFα treated mice (solid line, open squares, n = 6), as compared to the APC Δ468 (dotted line & closed triangles). (e) Frequencies of CD11b + Gr1 + amongst total intestine live MNCs; APC Δ468 Ctsb −/− intestine (light gray bar, 0.56±0.15%, P<0.001), anti-TNFα treated (dark gray bar, 0.71±0.22%, P<0.001), untreated APC Δ468 (open bar, 4.2±0.093%), wt control intestine (black bar, 0.15±0.051%). (f) Frequencies of CD11b + F4/80 + in the APC Δ468 (5.03±0.78%), APC Δ468 Ctsb −/− intestine (5.36±0.92%), and anti-TNFα treated intestine (dark gray bar, 3.0±0.67%).

Article Snippet: Both of these myeloid cell types contributed equally to the local activation of the ProSense 680 probe.

Techniques:

Journal: PLoS ONE

Article Title: Live Imaging of Cysteine-Cathepsin Activity Reveals Dynamics of Focal Inflammation, Angiogenesis, and Polyp Growth

doi: 10.1371/journal.pone.0002916

Figure Lengend Snippet: Spectral separation of images from, (a&b) APC Δ468 mice, (c&d) Ctsb −/− APC Δ468 , (e&f) anti-TNFα treated APC Δ468 mice; ProSense-680 (green; CathepsinB), AngioSense-750 (red; blood vessels). The objective used was the UplanApo 4× (pixel size 5.4 µm, lateral resolution 12.42 µm). (g) Mean particle volumes in the Z stacks of fluorochrome 680 (open bar: APC Δ468 , 120554±86906 µm; gray bar: APC Δ468 Ctsb −/− , 26609±5268 µm 3 ; anti-TNFα treated APC Δ468 , 8639±1570 µm 3 ; P<0.0001 one sample test). (h) Mean particle volumes in the Z stacks of fluorochrome 750 (open bar: APC Δ468 , 15501±2144 µm 3 ; gray bar: APC Δ468 Ctsb −/− , 6963±1236 µm 3 , anti-TNFα treated, 3700±1444 µm 3 ; P<0.0001 one sample test).

Article Snippet: Both of these myeloid cell types contributed equally to the local activation of the ProSense 680 probe.

Techniques:

Journal: Journal of Clinical Medicine

Article Title: Skeletal Muscle Subpopulation Rearrangements upon Rhabdomyosarcoma Development through Single-Cell Mass Cytometry

doi: 10.3390/jcm10040823

Figure Lengend Snippet: Generation of eRMS tumors in LSL-Kras G12D/+ ;Tp53 Fl/Fl mice. ( A ) Experimental workflow. LSL-Kras G12D/+ ;Tp53 Fl/Fl conditional mice were injected intramuscularly with Ad-Cre in order to induce genetic rearrangements, the constitutive activation of Kras and the inactivation of Tp53, which lead to eRMS tumor masses in a spatially and temporally controlled manner. ( B ) LSL-Kras G12D/+ ;Tp53 Fl/Fl conditional mice were injected with Ad-Cre and Ctrl vector. Tumor growth was assessed at three, five and seven weeks after Ad-Cre infection through FMT performed by intravitreal administration of the fluorescent probe (ProSense 680). This probe was activated by tumor cathepsin proteases to yield a fluorescence signal (black: low cathepsin activity; white: high cathepsin activity). ( C ) Representative H&E staining on histological sections from Ctrl and Ad-Cre infected muscles at three, five and seven weeks after Ad infection. ( D , E ) Immunofluorescence staining on sections of Ad-Cre infected muscles at three, five and seven weeks after infection. Sections were stained with antibodies raised against Caveolin-3 ( D ) and α-SMA ( E ). Scale bars: 100 µm.

Article Snippet: The emerging tumors were imaged by injecting a fluorescent probe (ProSense 680) that was specifically activated by cathepsin proteases, a tumor-specific enzymatic activity [ ].

Techniques: Injection, Activation Assay, Plasmid Preparation, Infection, Fluorescence, Activity Assay, Staining, Muscles, Immunofluorescence