Ab133021

Blood was collected from the heart of each mouse after fasting overnight. The supernatant was obtained after centrifugation as previously described [24 (link)]. PGE2 levels in both serum and serum-free primary osteocytes CM were detected using a commercially available kit (ab133021, Abcam, USA), according to the manufacturer’s protocols.

NP effect on the release of five inflammatory mediators in LPS+ RAW264.7 was further quantified using mouse TNF-α, IL-1β and IL-10 ELISA kits purchased from Sigma-Aldrich (RAB0477, RAB0274 and RAB0245); and mouse IL-6 and PGE₂ ELISA kits purchased from abcam (ab222503 and ab133021). In brief, cells were seeded into six well culture plates at a density of 3 × 10⁵ cells/well and cultured for 24 h, then, cells were activated with LPS in RAW264.7 culture medium (1 µg mL⁻¹ for IL-1β quantification and 500 ng mL⁻¹ for TNF-α, IL-6, PGE₂ and IL-10) and exposed simultaneously to NPs (CLX-10, TNX-5 and DEX-15 at 0.50 mg mL⁻¹). Following a 24 h incubation, supernatants were collected and stored at −20 °C until use. Levels of TNF-α, IL-1β, IL-6, PGE₂ and IL-10 in cell culture supernatants were determined by the corresponding ELISA kit according to the protocol recommended by the manufacturer. LPS-stimulated and unstimulated cells treated with PBS were used as controls (LPS+ and LPS-, respectively). Experiments were performed using five replicates per formulation and results were expressed as mean ± SD. ANOVA were performed comparing tested NPs and LPS+ at significance levels of p < 0.05, p < 0.005 and p < 0.001 and comparing NPs with each other at significance levels of p < 0.05, p < 0.005 and p < 0.001.

The concentrations of PGE₂ (ab133021, Abcam), SDF-1 (ab100637, Abcam), VEGF (ab100663, Abcam), IL-10 (ab100549, Abcam), and TGF-β1 (ab100647, Abcam) in culture media were measured using commercial ELISA kits according to the manufacturer's protocols.

PGE2 levels were determined using an ELISA kit (ab133021; Abcam, Cambridge, MA, USA), according to the manufacturer’s guidelines.

Muscle extract samples for ELISA were obtained by homogenizing quadriceps muscles of young and aged mice in homogenization buffer (0.1 M phosphate, pH 7.4, containing 1mM EDTA and 1uM indomethacin). Prostaglandin E2 ELISA was performed according the protocol provided by the manufacturer (Abcam, cat# ab133021).

To assess the anti-inflammatory effect of 10HA and 10HA 120NP HGs further, ELISA assays were performed to quantify the release of six pro-inflammatory mediators by LPS-stimulated RAW264.7. Mouse TNF-α, IL-6, IL-10, IL-12p40, IL-23, and PGE2 ELISA kits were purchased from abcam (ab208348, ab222503, ab255729, ab236717, ab119545, and ab133021, Cambridge UK). In brief, cells were seeded on top of the HGs and CGs at a density of 2 × 10⁶ cells/well and cultured for 24 h. Then, cells were exposed to medium containing LPS (500 ng/mL) and incubated overnight. Media were collected and stored at −20 °C until use. Levels of mouse TNF-α, IL-6, IL-10, IL-12p40, IL-23, and PGE2 in cell culture supernatants were determined by the corresponding ELISA kit according to the protocol recommended by the manufacturer. Experiments were performed using six replicates per formulation. ANOVA was performed at significance levels of p < 0.05, p < 0.01, and p < 0.001.