Calcein blue

During the experimental follow-up, three different calcium binding fluorophores were administered, by subcutaneous injection, to follow bone apposition and mineralization over time, which are to be detected in histological sections. Injection of Calcein Green (25 mg/kg, Fluka, Sigma Aldrich, Germany) was performed at 2 weeks and Xylenol Orange (30 mg/kg, Fluka, Sigma Aldrich, Germany) at 4 weeks, and Calcein Blue (25 mg/kg, Fluka, Sigma Aldrich, Germany) was injected on the day before sacrifice.
After sacrifice and sampling for bone culture, tibiae were fixated in 4% formaldehyde/PBS for 4 weeks and embedded in polymethylmethacrylate (PMMA) (Technovit 9100, Hereaus-Kulzer, Germany). After polymerization, sections were stained according to Masson-Goldner (Carl Roth, Germany) and Gram (without a safranin counterstain) and subsequently 50 μm sections were obtained using a saw microtome (SP 1600, Leica, Germany). Sections were analysed and digitized by light microscopy (Axioscope A1, AxioVision LE release 4.8.2, Carl Zeiss, Germany). The localization of calcium binding fluorophores in the bony tissue was visualized by fluorescence microscopy (Leica DMRB, Leica IM50 version 1.2 release 19, Leica, Germany) on unstained PMMA sections. Acquired images were merged using Photoshop CS3 (Adobe Systems, USA) to generate overview images.

Animals assigned to a 12-week implantation period received a series of four fluorochromes (SigmaAldrich; Munich, Germany) by subcutaneous injection with a 2-week interval, starting two weeks after implantation surgery: Calcein blue (product no. M1255, dose 30 mg/kg; week 2), calcein green (C0875, 10 mg/kg; week 4), alizarine complexone red (A3882, 25 mg/kg; week 6) and rolitetracycline yellow (R2253, 25 mg/kg; week 8). Before injection, the solutions were set to neutral pH (7.2–7.4), then filtered through a 0.22 sterile millipore filter, and finally checked for fluorescence.

Mice were intraperitoneally injected with 15 mg/kg of calcein dye (Sigma‐Aldrich) solution in Dulbecco's phosphate buffered saline on days −5 and −2 prior to harvest. Femurs were fixed, embedded in methyl methacrylate resin, and cut into 5‐μm sections. For dynamic histomorphometry, slides were counterstained with 1% Calcein Blue (Sigma‐Aldrich), and fluorescent intra‐label thickness was quantified. For histological assessment of the growth plate, slides were dual stained with Safranin O and Fast Green.

To assess cell-surface expression of CAR and SSTR2, transduced cells were stained with FITC anti-c-myc antibody (Miltenyi Biotec, clone SH1-26E7.1.3) and APC anti-human SSTR2 antibody (R&D systems, clone 402038). T lymphocytes and sub-populations were identified using a cocktail of anti-human PE-Cy5 CD3, anti-human PE CD4 and anti-human FITC CD8 antibodies (Biolegend, clone UCHT1; RPA-T4; RPA-T8). Live cells were selected using calcein blue (Sigma-Aldrich) staining along with forward- and side-scatter gating. Flow cytometry was performed on a Gallios flow cytometer (Beckman Coulter, Inc.) and analyzed with FlowJo software (Tree Star, Inc.).

Methylsulfonylmethane (MSM), Ascorbic acid, β-glycerophosphate, Calcein Blue, MTT assay kit, Alkaline phosphatase staining kit, Cystamine, and GAPDH antibody were purchased from Sigma (St. Louis, MO). The following antibodies were bought from the company indicated in parenthesis: Collagen alpha 1 (Col 1; Novus Biological; Littleton, CO); Osteopontin (OPN; Abcam, Cambridge, UK), Transglutaminase (TG2; Abcam, Cambridge, UK), Runt-related transcription factor 2 (RUNX2) and HRP conjugated (mouse or rabbit) secondary antibodies (Santa Cruz Biotechnology, Dallas, TX); Osterix (Millipore, MA, USA). Alizarin Red S (ARS) 2% staining solution was from LifeNet® Cell Technology (CM-0058; Fredrick, MD). Fluorochrome-conjugated secondary antibody Alexa Fluor 488 (#4412) and Alexa Fluor 555 (#8953) (Cell Signaling technology®, Danvers, USA) Super Signal^™ West Pico Chemiluminescent substrate was bought from Thermo Fisher Scientific (Waltham, Massachusetts). Demineralized and mineralized bone particles were from LifeNet Health^®(Virginia Beach, Virginia).

The rationales, protocols, and quantifications of bone remodeling imaging were detailed in our work published previously^{6 (link)}. In brief, two calcium-binding dyes were administered sequentially. The first calcium-binding dye (Dye 1, Calcein Blue, Sigma, 30 mg/kg) was administered intraperitoneally 48 h before imaging to label and track the change of bone fronts over the course of 2 days, which approximately represents one cycle of bone resorption. Calcein Blue was chosen to be spectrally compatible with Rhod-5N and AF488. Its effect was transient (Fig. 2d) and did not alter the serum calcium on the day of imaging as verified by the Arsenazo assay. The second calcium-binding dye to label all the bone fronts (Alizarin Red, 40 mg/kg) was injected on the day of imaging but after the acquisition of the Rhod5N/AF488 data so it does not interfere with the calcium measurement. As Dye 1 would be eroded if bone resorption has occurred, the Dye 1 to Dye 2 ratio in a single BM cavity (the concave endosteum) indicates the stage of bone remodelling during the 48 h period. We then defined bone cavities as (i) deposition type (D-type; dye 1:dye 2 > 75%); (ii) resorption type (R-type; dye 1:dye 2 < 25%), and (iii) mixed type (M-type; dye 1:dye 2 between 25 and 75%. For quantifying fractions of cavity types, 3D maps of calvaria were acquired and analyzed.