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  • br As previously describes Falk et al b


    As previously describes (Falk et al., 2015b), rats were allowed to move freely in a transparent standard cage without bedding (560 mm × 350 mm × 200 mm, Scanbur A/S, DK). Following 10 min of acclimation, the animal was observed for 3 min and assigned a limb use score from 3 to 0 as follows: 3: Normal use of leg, 2: mild or in-significant limping and normal body distribution, 1: significant limping and displacement of bodyweight, 0: significant limping and partly lack of use during sitting or locomotor activity.
    Rats were placed in the incapacitance tester (Linton Instrumentation) and allowed to acclimatize for 2–3 min. Measurements were performed over a CT99021 of 3 s and in triplicates. An average weight-bearing ratio was subsequently calculated as the amount of weight placed on the cancer-bearing leg divided by the total amount of weight placed on both legs and used for data analysis.
    2.5. Bioluminescence
    Rats were anesthetized with 2.5% isoflurane (Isobar Vet, Nomeco, Denmark) and i.p. injected with 40 mg/kg D-Luciferin in PBS (PerkinElmer, Denmark) and returned to the induction chamber. 10 min following injection the animal was moved to the imaging chamber and placed on their back (IVIS Lumina XR, Caliper Life Science, Belgium). Bioluminescent images were recorded in triplicated with binning M(4), F/stop 1 and exposure time from 1 to 60 s according to the power of the signal. Between each image the animal was re-positioned to minimized bias introduced by positioning in the chamber. Bioluminescence images were analyzed using IVIS Imaging software (Living Image©, version, Caliper Life Science, Belgium). For each image the radiance total flux measured as photons/sec was used following adjustment of the signal threshold to 5%.
    X-ray images (Binning: 1, F/Stop: 2, high resolution) were taken simultaneous with the bioluminescence images. X-ray images were calibrated to a standard aluminum wedge and processed in ImageJ by inverting the 18 bit image followed by an equalized histogram contrast enhancement. The mean gray value of a 349pixel ROI was quantified within the trabecular bone and calibrated to the average of two cor-responding background regions in the soft tissue proximate to the tibia.
    2.7. Drug administration
    AFC5261 (Affectis Pharmaceuticals AG, Germany) was dissolved in deionized water with 0.5% carboxymethylcellulose (Sigma, Denmark), 0.25% Tween20 (Sigma, Denmark). Morphine hydrochloride (mor-phine, Skanderborg Pharmacy, Denmark) was dissolved in 0.9% saline. All drugs were administrated by oral gavage. For chronic treatment animals were administrated twice a day from day 5 with 50, 100 or 300 mg/kg. Doses where chosen based on previous in-house experi-ments.
    Fluorometric Ca2+ assays in cell suspensions were performed in 384-microwell plates (Corning, USA) with a fluorescence-imaging
    microplate reader (POLARstar Omega, Germany). Briefly, suspensions of HEK293 cells (Fischer et al., 2016) stably expressing human, mouse and rat P2X7R, respectively, were incubated with the fluorescent in-dicator fluo-4/AM (4 μM, Invitrogen, Germany), in the dark for 30–45 min at 37 °C, centrifuged (100 ×g for 3 min), and re-suspended in HEPES-buffered solution (HBS), containing 133 mM NaCl, 4.8 mM KCl, 1.2 mM KH2PO4, 1.3 mM CaCl2, 1 mM MgCl2, 10 mM HEPES and 10 mM D-glucose adjusted to pH 7.4 with NaOH, or in a similar solution without MgCl2. To determine concentration-response relationships, test compounds were serially diluted down by a factor of two by using a programmable robotic liquid handling station (Freedom Evo 150, Switzerland). The maximal final concentration was 50 μM tanshinone IIA sulphonic sodium or 5 μM and 0.5 μM, respectively, depending on the compound and blocking potency at the corresponding kind of P2X7R. Microwell plates were repetitively scanned every 16 s in the fast scanning mode of the device. After 10 baseline cycles, ATP (in Mg2+-free HBS; final concentration of 1 mM) was injected into each well, and fluorescence intensities were followed after a delay of 2 min for up to 40 min (150 cycles) after ATP injection. Data were normalized to the baseline values before ATP application (F/F0).