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Osteosense 750ex

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OsteoSense 750EX is a fluorescent imaging agent designed for in vivo visualization and quantification of bone metabolism in small animal models. It is intended for use in preclinical research applications.

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Lab products found in correlation

Fmt 2500, by PerkinElmer (3 mentions) Iv100, by Olympus (2 mentions) Cm3050, by Leica (2 mentions) Truequant software, by PerkinElmer (2 mentions) Angiosense 680ex, by PerkinElmer (2 mentions) Cd31 alexa fluor 647, by BioLegend (1 mentions) Facsaria 2 cell sorter, by BD (1 mentions) Vibrant did cell labeling solution, by Thermo Fisher Scientific (1 mentions) Iv100 microscope, by Olympus (1 mentions) Sp dioc18 3, by Thermo Fisher Scientific (1 mentions) Celltracker cm dil, by Thermo Fisher Scientific (1 mentions) Rl 1102, by Vector Laboratories (1 mentions)

Spelling variants of this product

OsteoSense (4 citations) OsteoSense 750EX (NEV10053EX, (2 citations)

12 protocols using osteosense 750ex

1

In Vivo Bone and Vasculature Imaging

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To visualize bone structures, mice were administered intravenously with OsteoSense® 750EX (4 nmol/mouse, PerkinElmer). For in vivo endothelial cells labeling, mice were given i.v. APC anti-mouse CD31 (MEC13.3), Alexa Fluor® 647 anti-mouse VE-Cadherin (BV13) and APC anti-mouse Sca-1 (D7, 2 μg/mouse in 100μl PBS).
Courties G., Herisson F., Sager H.B., Heidt T., Ye Y., Wei Y., Sun Y., Severe N., Dutta P., Scharff J., Scadden D.T., Weissleder R., Swirski F.K., Moskowitz M.A, & Nahrendorf M. (2014). Ischemic Stroke Activates Hematopoietic Bone Marrow Stem Cells. Circulation research, 116(3), 407-417.
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2

Intravital Microscopy of Bone Marrow B Cells

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For serial intravital microscopy of the calvarium, bone marrow B cell precursors (Lineage− CD93+) from dsRed reporter mice and mature B cells from the spleen (CD19+ IgD+) were FACS-sorted using a FACSAria II cell sorter (BD). For B cell progenitor imaging experiments, 106 cells were injected i.v. into non-irradiated recipient C57BL/6 mice one day prior to stroke induction. To visualize mature B cell accumulation in the bone marrow, 5×106 cells were injected i.v. one week prior to surgeries. To highlight bone architecture, OsteoSense® 750EX, a fluorescent bisphosphonate imaging agent (Perkin Elmer), was administered i.v. 24 hours prior to imaging (4 nmol/mouse, PerkinElmer). To outline the vasculature, 15μg CD31-Alexa Fluor 647 (102516, BioLegend) 30 min prior to imaging. In vivo imaging was performed using a confocal microscope (IV100 Olympus). Z-stack images for each location were acquired at 2μm steps, and post-processing was performed using Image J software (NIH).
Courties G., Frodermann V., Honold L., Zheng Y., Herisson F., Schloss M.J., Sun Y., Presumey J., Severe N., Engblom C., Hulsmans M., Cremer S., Rohde D., Pittet M.J., Scadden D.T., Swirski F.K., Kim D.E., Moskowitz M.A, & Nahrendorf M. (2019). Glucocorticoids Regulate Bone Marrow B Lymphopoiesis After Stroke. Circulation research, 124(9), 1372-1385.
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3

Visualizing Bone and Vasculature in Mice

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To identify bone, animals were injected with OsteoSense® 750EX (4nmol/mouse, PerkinElmer) the day before imaging. The vasculature was labeled in vivo with 50 µl of RPE anti mouse CD31 antibody (#553373 BD Biosciences) or isolectin B4 (FL-1201 Vector labs), injected IV 1 hour before sacrifice. Mice were euthanized 2 hours after either skull or tibia microinjection and perfused with 20 ml of PBS, or 7 hours after systemic injection of evans blue 3% (w/vol in sterile saline). The skull and the tibiae were dissected and cleaned before fixation for 1 hour in 4% paraformaldehyde in PBS, then rinsed twice in PBS for 10 minutes. Tibiae were then embedded in Tissue-Tek® O.C.T( #4583 Sakura Finetek Inc Torrance CA USA) on dry ice, and kept at −20 Celsius. They were later shaved on a cryostat (CM 3050S, Leica, Wetzlar, Germany) to expose the bone marrow compartment. Z-stacks images were acquired with 2 µm steps at 20x magnification. Images were processed using the Imaris software ™ (Bitplane, Zurich, Switzerland). Maps of the entire bones for the injection sites were generated by the stitching of multiple images acquired at 4X magnification, using the Affinity Designer® software (Serif, Nottingham, UK). Size of the channels was determined manually using the measuring distance tool on Imaris software™ (Bitplane, Zurich, Switzerland).
Herisson F., Frodermann V., Courties G., Rohde D., Sun Y., Vandoorne K., Wojtkiewicz G.R., Masson G.S., Vinegoni C., Kim J., Kim D.E., Weissleder R., Swirski F.K., Moskowitz M.A, & Nahrendorf M. (2018). Direct vascular channels connect skull bone marrow and the brain surface enabling myeloid cell migration. Nature neuroscience, 21(9), 1209-1217.
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4

Tracking Donor Stem Cell Homing

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LKS cells were isolated from CD45.1STEM donor mice as described in the previous section. Freshly isolated LSK cells were labeled with a Vibrant™ DiD cell-labeling solution (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine perchlorate) (Thermo Fischer Scientific, V22887) for 15 min at 37degree in the dark according to manufacturer instructions. 3 × 104 LKS cells and 0.2 × 106 supportive bone marrow cells were transplanted to lethally irradiated (9.5Gy) recipient mice via retro-orbital injection. 48hours after LKS cells injection, mice were anesthetized by isoflurane inhalation with 2% O2. To visualize the bone structure, mice were previously administered intravenously with Osteosense® 750EX (4nmol/mouse, Perkin Elmer). To visualize the vasculature, mice were injected intravenously with anti-mouse CD31-AF647 (5μg/mouse), anti-mouse VE-Cadherin-AF647 (5μg/mouse) and anti-mouse Sca1-APC (2μg/mouse) antibodies (Key resource table) in 100ul of sterile PBS. The calvarium of the mice was imaged using an Olympus IV100 microscope (4X and 20X objective, Z-Stack of 2um). All images were analyzed and stitched using ImageJ software and Did+ cells were enumerated.
Severe N., Karabacak N.M., Gustafsson K., Baryawno N., Courties G., Kfoury Y., Kokkaliaris K.D., Rhee C., Lee D., Scadden E.W., Garcia-Robledo J.E., Brouse T., Nahrendorf M., Toner M, & Scadden D.T. (2019). Stress-induced changes in bone marrow stromal cell populations revealed through single-cell protein expression mapping. Cell stem cell, 25(4), 570-583.e7.
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5

Multicolor Imaging of Bone and Immune Cells

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Bone sections were stained with Chrna7 (ANC-007, Alomone Labs) and Sca1 (122502, clone E13–161.7, Biolegend); secondary antibodies anti-rat 488 (20027, Biotium) and anti-rabbit 555 (20038, Biotium). Imaging was performed on a Leica TCS SP8 confocal microscope. For confocal microscopy of the femur in ChatGFP mice, OsteoSense 750EX (NEV10053EX, 4 nmol per mouse, PerkinElmer) was administered i.v. to outline bone architecture, CD31-PE (102508, clone MEC13.3, 15 μg per mouse, BioLegend) and Sca1-PE (12–5981-83, clone D7, 15 μg per mouse, eBioscience) to outline vasculature and B220-APC (103212, clone RA3–6B2, 15 μg per mouse, BioLegend) to identify B cells. B220 isotype antibodies (400512, clone RTK2758, 15 μg per mouse, BioLegend) were used for control stainings. For selected experiments, the femur was optically cleared by incubating the fixed femur in a RapiClear (SunJin Lab) solution and imaged using a confocal microscope (IV100 Olympus).
Schloss M.J., Hulsmans M., Rohde D., Lee I.H., Severe N., Foy B.H., Pulous F.E., Zhang S., Kokkaliaris K.D., Frodermann V., Courties G., Yang C., Iwamoto Y., Knudsen A.S., McAlpine C.S., Yamazoe M., Schmidt S.P., Wojtkiewicz G.R., Masson G.S., Gustafsson K., Capen D., Brown D., Higgins J.M., Scadden D.T., Libby P., Swirski F.K., Naxerova K, & Nahrendorf M. (2022). B lymphocyte-derived acetylcholine limits steady-state and emergency hematopoiesis. Nature immunology, 23(4), 605-618.
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6

In vivo Osteogenesis and Angiogenesis Evaluation

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In order to functionally evaluate the occurrence of osteogenesis and angiogenesis within the scaffold in a time-course manner, some animals were analysed by in vivo FMT (FMT 2500, Perkin Elmer, Monza, Italy). In particular, all implanted animals, as well as four unoperated mice, received an injection of 100 μl of OsteoSense 750EX (Perkin Elmer), plus 100 μl of AngioSense 680EX (Perkin Elmer) into the tail vein one day before sacrifice. These fluorescent probes specifically bind to newly formed hydroxyapatite and endothelial cells, respectively. Twenty-four hours after the probe injection, FMT images were acquired. During the acquisition, animals were kept under isoflurane anaesthesia and placed into the glass FMT imaging cassette. Acquisition and analysis of FMT images were carried out by using the TrueQuant software (Perkin Elmer). For analysis, the region of interest (ROI) was selected and the extent of osteogenesis or angiogenesis was quantified by measuring the amount of fluorescence probe (in pmol) into the ROI after choosing a concentration threshold. This threshold has been determined by keeping the volume of ROI constant (50 mm3). Animals were sacrificed immediately after imaging.
Calabrese G., Giuffrida R., Forte S., Fabbi C., Figallo E., Salvatorelli L., Memeo L., Parenti R., Gulisano M, & Gulino R. (2017). Human adipose-derived mesenchymal stem cells seeded into a collagen-hydroxyapatite scaffold promote bone augmentation after implantation in the mouse. Scientific Reports, 7, 7110.
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7

Aortic Calcification Imaging with Fluorescent Agent

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Aortic calcification was detected by a bisphosphonate derivative near-infrared fluorescent imaging agent (Osteosense 750 EX, PerkinElmer, Boston, MA, USA), which was injected into the rats through the tail vein 24 hours before imaging. The rats were euthanized, and the aorta was perfused with normal saline. After dissection, FRI was used to map the near-infrared fluorescence signal using Osteosense 750 EX fluorescent imaging agent (excitation/emission: 745/800 nm).
Yan J., Xiao H., Zhou X., Li Y., Zhao S., Zhao X., Liu Y., Liu M., Xue F., Zhang Q., Zhao W., Li L., Su Y, & Zeng W. (2023). Engineered exosomes reprogram Gli1+ cells in vivo to prevent calcification of vascular grafts and autologous pathological vessels. Science Advances, 9(29), eadf7858.
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8

Visualizing Bone and Vasculature in Mice

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To identify bone, animals were injected with OsteoSense® 750EX (4nmol/mouse, PerkinElmer) the day before imaging. The vasculature was labeled in vivo with 50 µl of RPE anti mouse CD31 antibody (#553373 BD Biosciences) or isolectin B4 (FL-1201 Vector labs), injected IV 1 hour before sacrifice. Mice were euthanized 2 hours after either skull or tibia microinjection and perfused with 20 ml of PBS, or 7 hours after systemic injection of evans blue 3% (w/vol in sterile saline). The skull and the tibiae were dissected and cleaned before fixation for 1 hour in 4% paraformaldehyde in PBS, then rinsed twice in PBS for 10 minutes. Tibiae were then embedded in Tissue-Tek® O.C.T( #4583 Sakura Finetek Inc Torrance CA USA) on dry ice, and kept at −20 Celsius. They were later shaved on a cryostat (CM 3050S, Leica, Wetzlar, Germany) to expose the bone marrow compartment. Z-stacks images were acquired with 2 µm steps at 20x magnification. Images were processed using the Imaris software ™ (Bitplane, Zurich, Switzerland). Maps of the entire bones for the injection sites were generated by the stitching of multiple images acquired at 4X magnification, using the Affinity Designer® software (Serif, Nottingham, UK). Size of the channels was determined manually using the measuring distance tool on Imaris software™ (Bitplane, Zurich, Switzerland).
Herisson F., Frodermann V., Courties G., Rohde D., Sun Y., Vandoorne K., Wojtkiewicz G.R., Masson G.S., Vinegoni C., Kim J., Kim D.E., Weissleder R., Swirski F.K., Moskowitz M.A, & Nahrendorf M. (2018). Direct vascular channels connect skull bone marrow and the brain surface enabling myeloid cell migration. Nature neuroscience, 21(9), 1209-1217.
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9

Longitudinal Osteogenesis and Angiogenesis Imaging

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In order to functionally assess the time-course of osteogenesis and angiogenesis within the implanted scaffolds, animals were analyzed by FMT (FMT 2500, Perkin Elmer). In particular, the implanted animals as well as four intact mice, received an injection of 100 μl of OsteoSense 750EX plus 100 μl of AngioSense 680EX (Perkin Elmer) into the tail vein, according to the manufacturer’s instructions. Twenty-four hours later, FMT scans were performed. During the acquisition, animals were kept under isoflurane anesthesia. Acquisition and data analysis were carried out using the TrueQuant software (Perkin Elmer). For analysis, the region of interest (ROI) was selected and the extent of osteogenesis or angiogenesis was quantified by measuring the amount of fluorescence probe (in pmol) into the ROI after choosing a concentration threshold. This threshold has been determined by keeping the volume of ROI constant (50 mm3). The animals were sacrificed immediately after imaging.
Calabrese G., Giuffrida R., Forte S., Salvatorelli L., Fabbi C., Figallo E., Gulisano M., Parenti R., Magro G., Colarossi C., Memeo L, & Gulino R. (2016). Bone augmentation after ectopic implantation of a cell-free collagen-hydroxyapatite scaffold in the mouse. Scientific Reports, 6, 36399.
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10

In Vivo Fluorescence Imaging of Mice

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Mice (n=4) were injected with 4nmol/g Osteosense 750EX (Perkin Elmer Inc, Boston, MA) intravenously. This dose was chosen based on the recommendations of the manufacturer. Approximately 24 hours later, the mice were anesthetized with 2% isofluorane and positioned in an FMT 2500 (Perkin Elmer Inc., Boston, MA). Scanning was performed using the 750EX channel. The entire mouse, except head and tail, were in the field-of-view. The scan lasted approximately 20 min. The images were reconstructed using the on-board TrueQuant software into a 512 × 512 × 13 array with a voxel size of 0.163 × 0.163 × 1 mm3.
Felix D.D., Gore J.C., Yankeelov T.E., Peterson T.E., Barnes S., Whisenant J., Weis J., Shoukouhi S., Virostko J., Nickels M., McIntyre J.O., Sanders M., Abramson V, & Tantawy M.N. (2014). Detection of breast cancer microcalcification using 99mTc-MDP SPECT or Osteosense 750EX FMT imaging. Nuclear medicine and biology, 42(3), 269-273.
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