Thermo scientific multiscan go

As the tibias from all animals were homogenized to measure primary outcome, we included serological biomarker measurement to address whether HBOT would affect either osteoclast or osteoblast activity. Plasma was separated from whole blood. Tartrate-resistant acid phosphatase 5b (TRAP 5b), a bone resorption marker specific to osteoclasts, was determined using a mouse TRAP 5b enzyme-linked immunosorbent assay [37 (link)]. Procollagen type 1 amino-terminal propeptide (P1NP), a marker of bone formation, was determined using an enzyme immunoassay [37 (link)]. Assays were performed in accordance with the manufacturer’s recommendations (Immunodiagnostic Systems Nordic, Copenhagen, Denmark) and analysed on a Thermo Scientific Multiscan Go (Thermo Fisher Scientific, Waltham, MA, USA) microplate spectrophotometer.

The sulfhydryl groups (SH) [51 ] were measured in the skeletal muscles homogenates. Briefly, 20 μL of homogenate (5%) was added to 200 μL of buffer (10 mM potassium phosphate buffer, pH 8.0). Next 30 μL of SDS (10 %) and 30 μL of DTNB (1 mM) were added. The measurements were conducted in duplicate. After this, the probes were mixed and incubated for 30 min in 37 °C. The absorbance was measured at 412 nm in microplate reader Thermo Scientific Multiscan Go (ThermoFisher Scientific, Vartaa, Finland). The values of the sulfhydryl groups (mmol/g of tissue) were calculated against sample blank (without DTNB) from the standard curve of reduced glutathione.

Supernatants were obtained from iMϕ and MDM cultures, aliquoted and stored at −20°C until the day of analysis. Cytokines were determined using MILLIPLEX MAP Human Cytokine/Chemokine kit (cat #HCYTOMAG-60K, EMD Millipore Corp., MA, USA) that evaluates the following 41 factors: EGF, CCL11 (eotaxin), CSF2 (GM-CSF), CSF3 (G-CSF), IFNα2, IFNγ, IL-10, IL-12P40, IL-12P70, IL-13, IL-15, IL-17A, IL-1RA, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, CXCL8 (IL-8), CXCL10 (IP-10), CCL2 (MCP-1), CCL3 (MIP-1α), CCL4 (MIP-1β), CCL5 (RANTES), TNFα, TNFβ, VEGF, FGF-2, TGF-α, FLT-3L, CXC3CL1 (fractalkine), CXCL1 (GRO1), CCL7 (MCP-3), CCL22 (MDC), PDGF-AA, PDGF-AB/BB, sCD40L, and IL-9. All the procedures were done in accordance with the manufacturer's recommendations.
To examine iMϕ reactivity to inflammatory stimuli, cells were stimulated with E. coli LPS (100 ng/ml, cat #L4391, Sigma Aldrich) and/or IFN-γ (20 ng/ml) for 24 h at 37°C. Culture supernatants were analyzed using Human TNF-α ELISA assay (cat # P3H 2017/5961, Vector-Best, Novosibirsk, Russian Federation) according to the manufacturer's instructions using microplate spectrophotometer Thermo Scientific™ Multiscan™ GO and ScanIt™ Software (Thermo Fisher Scientific, Waltham, Ma, USA).