Osteosense 680ex

HAoSMCs were incubated overnight at 37 °C with OsteoSense 680EX (1:250, Perkin Elmer, Traiskirchen, Austria) [42 (link)]. Images were acquired with the ChemiDoc MP imaging system (Bio-Rad Laboratories, Vienna, Austria) with excitation/emission (bandpass) wavelengths of 680/715(30) nm [43 (link)]. HAoSMCs were decalcified in 0.6 M HCl at 4 °C overnight and the calcium content was quantified with the QuantiChrom Calcium assay kit (BioAssay Systems, Hayward, CA, USA) [14 (link),21 (link),37 (link)]. Proteins were isolated using 0.1 M NaOH/0.1% SDS buffer and quantified by the Bradford assay (Bio-Rad Laboratories, Vienna, Austria). Data are shown normalized to total protein concentration.

In vivo bone growth were imaged and analyzed with two commercial fluorescent in vivo bisphosphonate imaging probe OsteoSense^® 680 Ex (OS680) and OstenSense^® 800 (OS800) (PerkinElmer, MA, USA) with two distinct wavelengths modified from suggested protocol and previous study [23 (link)]. Briefly, anesthetized mice were injected retro-orbitally with dissolved fluorescent probes (10 nmol/kg) 48 h before imaging. Hair was removed from the hind legs. Florescent signals and X-ray images were captured by combination of multispectral fluorescence and digital radiograph function of in vivo Multispectral FX PRO system. After anesthesia, signals of injected Os680 were detected in vivo with excitation wavelength at 650 nm and emission at 700 nm at week 2, while Os800 was injected and detected (excitation 760 nm/emission 830 nm) at week 3. At week 4, both florescent signal of Os650 and Os800 in both sides of femur were measured ex vivo after femurs were harvested. Florescent and radiographic images obtained were overlayed and analyzed with Carestream MI Software. ROIs of same area were selected from operated and contralateral femurs versus surrounding skin (as background signal). Os signal intensity of fractured right femurs was normalized by intensity of contralateral side.

OsteoSense dye (OsteoSense 680 EX and NEV10020EX; PerkinElmer, MA, United States) and near-infrared imaging were used to evaluate aorta calcification in mice as previously described (Malhotra et al., 2019 (link)). Mice (Ctrl, CKD, and CKD + DPD; n = 5/group) were anesthetized with isoflurane and injected with 2 nmol OsteoSense dye dissolved in 100 µL DPBS retro-orbitally. After 24 hours, mice were killed by CO₂ inhalation; the mice were perfused with 5 ml of ice-cold PBS, and aortas were isolated, cleaned, and analyzed ex vivo by an IVIS Spectrum In Vivo Imaging System (PerkinElmer, MA, United States). The observer who performed this measurement was blinded to the group assignment.

Mice were injected IV with 4 mg of ArthritoMab monoclonal antibody cocktail (MD Biosciences), and then IP 3 days later with 100 μg LPS (MD Biosciences). In some experiments, 0.7 mg of anti-MICL or isotype control antibodies were administered IP every 24 h from day 4 to day 13. Daily clinical scoring was undertaken using a scale of 0 (no visible signs of redness or swelling) to 4 (extensive swelling with signs of deformity). To generate radiation chimeras, animals received 2×500 rad (IBL437C irradiator, Cis Bio International) and were then reconstituted with 2–4×10⁶ bone marrow cells from donor mice. For histology, joints were isolated, fixed, decalcified, sectioned and stained with H&E. In vivo imaging was performed using a Bruker in vivo MS FX Pro imager, after a single IV administration of OsteoSense 680EX (NEV10020EX, PerkinElmer, UK), 24 h before the start of the collagen antibody-induced arthritis (CAIA) protocol. Twenty-four hours before image acquisition, animals were also injected IV with ProSense 750EX (NEV10001EX, PerkinElmer, UK).

In addition, the aortic valve peak flow velocity and aortic valve mean gradient were measured in the apical five-chamber view by continuous wave doppler in systole. The presence or absence of aortic regurgitation was visualized in this five-cavity view by color Doppler. Furthermore, aortic stenosis was estimated in two-dimensional parasternal long-axis view by measuring the aortic valve cusp separation in systole.
At sacrifice, the heart was harvested and weighed, and a section of the left ventricle was snap-frozen for subsequent determination of LV fibrosis, using 8 µm thick histological slices stained with Sirius Red as previously described [12 (link)]. In addition, the aortic valve was carefully dissected and incubated over 24 h at 4 °C in a solution of a 20 nM Osteosense 680Ex^® (Perkin-Elmer, Waltham, MS, USA) in PBS. This fluorescent probe binds to hydroxyapatite with high affinity and thus allows for the detection of microcalcifications. Then, the valve was rinsed and snap-frozen for subsequent analysis using 8 µm thick histological slices with mounting medium containing DAPI. Pictures were acquired on an epifluorescence microscope (Axio Imager 1, Zeiss, Jena, Germany) equipped with an apotome using the Cyanine 5.5 filter for calcification detection and a DAPI filter for cell nuclei detection.