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Osteosense

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OsteoSense is a fluorescent imaging agent used for the visualization and quantification of bone-related processes in pre-clinical research. It is designed to bind to hydroxyapatite, a key component of bone, allowing for the non-invasive monitoring of bone metabolism and structure.

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Lab products found in correlation

Lsm 780 confocal microscope, by Zeiss (3 mentions) Apc anti mouse ly6g, by BioLegend (2 mentions) Rpe anti mouse cd31, by BD (2 mentions) F3506, by Merck Group (2 mentions) Atc2000, by World Precision Instruments (2 mentions) Formyl methionyl leucyl phenylalanine (fmlp), by Merck Group (2 mentions) Neofluar objective, by Zeiss (1 mentions) Macrophage colony stimulating factor, by Merck Group (1 mentions) Vitamin d, by Merck Group (1 mentions) Celltracker, by Thermo Fisher Scientific (1 mentions)

4 protocols using osteosense

1

Bone Microstructure Visualization

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Fixed and de-waxed paraffin embedded sections, 20 μm thick, with crystalline deposits were stained with 20 μM bone-tag 680RD (926-09374; Li-Cor) or 20 μM OsteoSense (NEV10020EX; Perkin Elmer) in aqueous buffer for 20 minutes at room temperature. Samples were imaged using a Zeiss LSM700 confocal microscope through a 63×/1.2 NA Zeiss Neofluar objective with an excitation wavelength of 620 nm and emission wavelength of 680 nm.
Pilgrim M.G., Lengyel I., Lanzirotti A., Newville M., Fearn S., Emri E., Knowles J.C., Messinger J.D., Read R.W., Guidry C, & Curcio C.A. (2017). Subretinal Pigment Epithelial Deposition of Drusen Components Including Hydroxyapatite in a Primary Cell Culture Model. Investigative Ophthalmology & Visual Science, 58(2), 708-719.
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2

Visualizing Skull Channel Cell Traffic

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To explore cell traffic through the skull channels to the meninges, we employed an organ bath system. After in vivo staining of the bone surface with OsteoSense® 750EX (4 nmol/mouse, PerkinElmer) the day before the experiment, the vasculature and neutrophils were stained with IV injections of 50 µl of RPE anti mouse CD31 (#553373 BD Biosciences) and 50 µl of APC anti mouse Ly6G (#127614 Biolegends) antibodies 1 hour before harvest. Using surgical scissors, we obtained a skull piece of about 4 by 4 mm, respecting the integrity of the dura and the visible edges of the marrow cavities. This specimen was flipped upside down and rapidly transferred into the organ bath. The specimen was placed on a stage and immersed with a solution of 50 µmolar fMLP (F3506 Sigma-Aldrich), used as chemoattractant, in HBSS. This protocol was adopted from previously published in vitro neutrophil migration assays35 (link). The organ bath was maintained at 37°C through a warming plate (ATC-2000 World Precision Instruments). The same conditions were implemented to explore neutrophil migration through the channels at 4–6 hours after permanent MCAO or after intracisternal injection of carrageenan, and corresponding sham or naive controls. Neutrophils exiting the channels were identified and counted using Imaris software™ (Bitplane).
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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3

Nanocomposite Scaffold Triculture Analysis

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A nanocrystalline hydroxyapatite (nHA)–poly(ester urethane) (PEUR) composite foam scaffold, fabricated as previously described,18 was cut to a size of 5 mm by 5 mm by 2 mm and wedged into a perfusion device consisting of a single channel with dimensions of 50 mm by 5 mm by 2 mm. Human MSCs, PBMCs, and MDA-MB-231 cells were labeled with fluorescent CellTracker™ (Thermo Fisher Scientific) membrane dyes (blue, green, and deep red, respectively) and seeded within the scaffold. The scaffold was cultured for 7 days under static conditions in α-MEM media containing 50 μg mL−1l-ascorbic acid, 10 nM dexamethasone, 10 mM β-glycerophosphate, 10 nM vitamin D, 25 ng mL−1 macrophage colony-stimulating factor, and 50 ng mL−1 receptor activator of nuclear factor kappa-B ligand (Sigma Aldrich). The scaffold was then perfused at 56 μL per second for 28 days in the same medium. The fluorescently-labeled triculture was imaged periodically with a Zeiss LSM 780 confocal microscope for 14 days after which the CellTracker™ dyes were no longer visible. At defined time points, 1 μM of PerkinElmer® OsteoSense® fluorescently labeled bisphosphonate was added to the perfused media to visualize mineralization.
O'Grady B.J., Geuy M.D., Kim H., Balotin K.M., Allchin E.R., Florian D.C., Bute N.N., Scott T.E., Lowen G.B., Fricker C.M., Fitzgerald M.L., Guelcher S.A., Wikswo J.P., Bellan L.M, & Lippmann E.S. (2021). Rapid prototyping of cell culture microdevices using parylene-coated 3D prints. Lab on a chip, 21(24), 4814-4822.
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4

Visualizing Skull Channel Cell Traffic

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To explore cell traffic through the skull channels to the meninges, we employed an organ bath system. After in vivo staining of the bone surface with OsteoSense® 750EX (4 nmol/mouse, PerkinElmer) the day before the experiment, the vasculature and neutrophils were stained with IV injections of 50 µl of RPE anti mouse CD31 (#553373 BD Biosciences) and 50 µl of APC anti mouse Ly6G (#127614 Biolegends) antibodies 1 hour before harvest. Using surgical scissors, we obtained a skull piece of about 4 by 4 mm, respecting the integrity of the dura and the visible edges of the marrow cavities. This specimen was flipped upside down and rapidly transferred into the organ bath. The specimen was placed on a stage and immersed with a solution of 50 µmolar fMLP (F3506 Sigma-Aldrich), used as chemoattractant, in HBSS. This protocol was adopted from previously published in vitro neutrophil migration assays35 (link). The organ bath was maintained at 37°C through a warming plate (ATC-2000 World Precision Instruments). The same conditions were implemented to explore neutrophil migration through the channels at 4–6 hours after permanent MCAO or after intracisternal injection of carrageenan, and corresponding sham or naive controls. Neutrophils exiting the channels were identified and counted using Imaris software™ (Bitplane).
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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