Optimax

Cytotoxycity was assessed by WST-1 assay according to the manufacturer’s instructions. After 48 hours 2.5 × 10⁴ Ba/F3 cells were exposed for 24 hours to increasing concentrations of cytarabine (Sigma-Aldrich, St Louis, MO, USA) or sorafenib (LTK Laboratories, St. Paul, MN, USA). 20 μl of WST-1 reagent were then added for a further incubation period of 3 hours. The ELISA reader (OptiMax, Molecular Devices, Sunnyvale, CA, USA) was set at a wavelength of 450 nm with a reference wavelength of 690 nm. Viability of cells after drug exposure was calculated as a percentage relative to untreated controls.

As previously described [11 (link)], venous blood was drawn from individuals undergoing outpatient evaluation at the Johns Hopkins A-T clinic between 2012 and 2014. Subjects were at baseline health and were not acutely ill at the time of their blood draws. The sera were separated and frozen at -80 C until analyzed. Concentrations of IL-6 and IL-8 were determined using commercially available EIA kits (R& D Systems, Minneapolis, MN). The optical density of each sample was determined using a microplate reader set to 450nm (Optimax, Molecular Devices, Sunnyvale, CA). Data was calculated from a standard curve and the results reported in pictograms of cytokine protein per milliliter for each cytokine. The mean minimum detectable dose for IL-6 was 0.7 pg/mL and for IL-8 was 3.5 pg/mL. Samples were assayed in duplicate and values were expressed as means +/- SD. All sample testing was performed in a masked fashion.

In the TRAP activity assay, BM cells were plated in 96-well plates at a density of 6 × 10⁴ cells per well and cultured in AMEM medium (GIBCO) containing 10% fetal bovine serum supplemented with 40 ng/ml RANKL (Peprotech) and 25 ng/ml M-CSF (R&D Systems) for 4 days. Then, cells were lysed in ice-cold phosphate buffer containing 0.1% Triton X-100. Lysates were added to 96-well plates containing phosphatase substrate (p-nitrophenol phosphate) and 40 mM tartrate acid buffer and incubated at 37°C for 30 min. The reaction was then stopped with the addition of 0.5 N NaOH. Absorbance was measured at 405 nm on a micro-plate reader (Optimax, Molecular Devices). TRAP activity was normalized to total protein which was determined using the Bradford assay (Bio-Rad).

The integrity of BBB was evaluated with a modified Evans blue extravasation method before sacrifice [23 (link)]. Briefly, 70 h post-ICH, rats were anesthetized with sodium pentobarbital (50 mg/kg, i.p.) and infused via the right femoral vein with 37 °C Evans blue dye (2% in 0.9% normal saline, 4 ml/kg) over 5 min. Two hours later, the rats were perfused with 300 ml normal saline to wash out any remaining dye in the blood vessels, and then the brains were removed and sectioned to 2 mm thickness with a rodent brain matrix. Coronal brain sections were taken starting at +2 mm and ending at −2 mm from bregma. BBB permeability was evaluated in the ipsilateral and contralateral striatum, and cerebellum. The cerebellum was used as an internal control. Each portion was weighed immediately and placed in 1 ml of 0.9% normal saline for homogenization of the sample. For protein precipitation, 1 ml of 60% trichloroacetic acid solution was added and vortexed for 2 min. Subsequently, the mixture was cooled for 30 min and centrifuged (1500 × g at 4 °C) for another 30 min. The absorbance of Evans blue in the supernatant was then measured with a spectrophotometer (Molecular Devices OptiMax, USA) at 610 nm. Dye concentration was expressed as μg/g of tissue weight and calculated from a standard curve obtained from known amounts of dye.

Cell viability was evaluated in preosteoclasts (bone-marrow-derived macrophages, BMMs) using the MTT assay as previously described [29 (link)]. Briefly, cells were seeded at a density of 10⁵ cells/well in 96-well plates and incubated with all tested compounds for 48 h in aMEM containing 10% fetal bovine serum supplemented with 25 ng/mL M-CSF (R&D Systems). After removal of the medium, each well was incubated with 0.5 mg/mL MTT (Sigma-Aldrich) in aMEM serum-free medium at 37 °C for 2 h. Upon removal of the medium, 200 μL of DMSO was added and the absorbance was measured at 550 nm on a microplate reader (Optimax, Molecular Devices). LC₅₀ values (mean ± SD calculated from five or more measuring points) were determined from three independent experiments.

Cells were cultured at 37 °C and labeled with 10 μl of 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide MTT (Sigma) at 3 mg/ml for 2 h. The precipitate was solubilized overnight with 10% SDS in 0.01-m HCl. Absorbance at 562 nm was recorded using an OPTImax microplate reader (Molecular Devices, Winnersh, UK).