Vector blue substrate kit

Alkaline phosphatase staining was performed with the Vector Blue Substrate kit (SK-5300) from Vector Laboratories. Briefly, iPSCs were fixed with 4% paraformaldehyde (PFA) supplemented with 0.1% Triton X-100 for 10 min. After rinses with PBS and H₂O, cells were then incubated with freshly prepared AP working solution in the dark for 40 min. Karyotype analysis of iPSCs was performed at WiCell Research Institute.

Alkaline phosphatase staining was monitored using a Vector Blue substrate kit (procedure number SK-5300, Vector Laboratories). According to the protocol, MC3T3-E1 or primary osteoblasts were incubated with the substrate working solution for 20–30 min. The whole procedure was protected from light.

Femurs were fixed in 4% paraformaldehyde for 3 days at 4 °C, incubated in 30% sucrose/PBS overnight, intramedullary pins were removed, and the femurs were embedded in Cryomatrix. 7 μm cryosections were collected using a tape transfer system (Section-lab, Japan)⁴⁹ . Callus area, cartilage area within the callus and mineralization was determined by Safranin O and von Kossa staining as previously describe in ref. ^{47 (link)}. TRAP staining was performed on 21-day fractures with Leukocyte acid phosphatase kit according to the manufacturer’s protocol and sections evaluated using OsteoMeasure software (OsteoMetrics Inc., Atlanta, GA, USA). A minimum of two sections were stained, scanned using Axioscan microscope (Zeiss, Germany) and analyzed with ImageJ software (NIH, USA). Osterix staining was perform as previously published in ref. ³ . To detect alkaline phosphatase Vector® Blue Substrate Kit (SK-5300, Vector laboratories) was used with 7 min of incubation time.

Alkaline phosphatase staining was monitored using a Vector Blue Substrate Kit (SK-5300; Vector Laboratories). According to the protocol, MC3T3-E1 or primary osteoblast cells were incubated with the substrate working solution for 20–30 min. The whole procedure was protected from light. After 2 min of rinsing in deionized water, slides were treated with Mayer’s hematoxylin solution for 10 min.
Cells were fixed in 70% ice-cold ethanol for 15 min and rinsed with double-distilled H₂O. Cells were stained with 40 mM Alizarin red S (Sigma), pH 4.0, for 15 min with gentle agitation. Cells were rinsed five times with double-distilled H₂O and then rinsed for 15 min using 1 × PBS with gently agitating.

MAIPS4510 ELISpot plates (Millipore) were coated with either 100 HA units of PR8, or 0.5 μg of goat anti-mouse unlabeled human adsorbed anti-IgM or -IgG (Southern Biotech) overnight at 4°C. Plates were blocked with media supplemented with FBS and washed with 1X PBS-TWEEN20 (HyClone). 1.5–3 x 10⁶ splenocytes (prepared as for FACS analysis, above) were added to the plate followed by serial 3-fold dilutions. Plates were incubated overnight at 37°C. Biotinylated anti-mouse IgG or IgM (Southern Biotech) was added at 1:1000 dilution followed by alkaline phosphatase-streptavidin (Southern Biotech) at 1:1000 dilution. ELISpot plates were developed with Vector Blue Substrate Kit (Vector Laboratories). Spots were counted using an ImmunoSpot Analyzer (CTL).

In vitro osteoclast differentiation was accomplished as previously described 38 (link). Briefly, BMM cells from femurs of 6 to 8 weeks old mice were cultured in full α-MEM with M-CSF (10 ng/mL, R&D systems, USA) for 1 d and then were differentiated into osteoclasts using RANKL (50 ng/mL, R&D systems, USA) and M-CSF (30 ng/mL) for 5 d. 50 μg/mL exosomes were added to 2 × 10⁶ recipient cells (TRAP⁺ monocytes) on the fourth day. For in vivo treatment, 50 μg exosomes were intravenously injected into 6-week-old female BALB/c nude mice every other day for 3 weeks. In the control group, PBS was used.
For the culture of osteoblast progenitor cells, calvariae from newborn mice were dissected aseptically and treated with 0.1% collagenase and 0.2% dispase. The cells were maintained in the minimum essential medium (MEM) alpha containing 10% FBS. For osteoblast differentiation, primary osteoblasts were cultured in α-MEM containing 10% FBS and 10 nM dexamethasone (Sigma), 50 µg/mL of ascorbic acid, and 5 mM β-glycerophosphate for 6 days. For in vitro treatment, 50 μg/mL of exosomes were added to 1 × 10⁴ recipient cells on the first day. Alkaline phosphatase (ALP) staining was carried out using a Vector Blue Substrate Kit (SK-5300; Vector Laboratories).