Spectramax 190 plate reader

Cells proliferation was determined using the CellTiter 96®
AQ_ueous One Solution Cell Proliferation Assay (Promega) according
to the manufacturer’s instructions. Briefly, the cells were incubated
with tetrazolium compound
[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,
inner salt for 1 h and the quantity of the formazan product was measured as the
absorbance at 490 nm on a SpectraMax 190 plate reader (Molecular Devices).
Apoptosis was assessed using the RealTime-Glo™ Annexin V Apoptosis Assay
(Promega) according to the manufacturer’s instructions. Luminescence over
30 min was read on a SpectraMax i3x Multi-Mode plate reader (Molecular Devices).
Data are reported at the maximal velocity (Vmax).

Alkaline phosphatase activity was measured using the Alkaline
Phosphatase Assay kit (colorimetric)(abcam). Approximately 5 ×
10⁵ cells were homogenized in assay buffer and centrifuged at
15,000 rpm for 15 min at 4°C. The supernatant was incubated with
p-nitrophenyl phosphate as a phosphatase substrate for 1 h at 25°C.
Dephosphorylation of the substrate was determined by reading the absorbance
at 405 nm on a SpectraMax 190 plate reader (Molecular Devices).

1800 cells/well were seeded onto white flat bottom half area 96-well plates (Greiner Bio-One, Kremsmuenster, AT) in 50 μl of growth medium and allowed to attach in an incubator for 24 h. Then the drugs in DMSO and/or DMSO vehicle controls as 11X solutions in growth medium were added to a total volume of 55 μl and 0.1% DMSO final. The cells were grown in an incubator for another 72 h. Then, 55 μl of CellTiter-Glo 2.0 (Promega, Madison, WI) reagent/well were added according to the manufacturer’s instructions and luminescence was quantified with a SpectraMax 190 plate reader (Molecular Devices, San Jose, CA). The CHEK1/2 inhibitor AZD7762 (Selleckchem, Houston, TX) and the NUAK1/2 inhibitor WZ4003 (Tocris Bioscience, Minneapolis, MN) were applied at 8 different concentrations between 10 μM and 4.6 nM (3-fold dilution steps) and Dinaciclib (Selleckchem) was applied at 8 different concentrations between 1 μM and 0.5 nM (3-fold dilution steps). Experiments were performed in four biological replicates. Growth inhibition curves were fitted using the GraphPad Prism software package (V5.0a) with a least-squares nonlinear regression model for curve fitting (One site - Fit logIC50 function).

1 μg of BMP-2 dissolved in 4mM HCl was added to 10 ml (BMP-2 concentration of 100 ng/ml) of 2% (w/v) nHA/CS solutions and mixed by stirring for 5 min at 0°C. Then nHA/CS/BMP-2 solutions were dripped into crosslinking medium and stirred at 300 rpm. After 4 h, nHA/CS/BMP-2 beads were separated and lyophilized using similar conditions as described earlier.
To study the release of BMP-2 from lyophilized particles, 10 mg of nHA/CS/BMP-2 particles were immersed in 2 ml of PBS in a glass vials and incubated at 37°C on an orbital shaker at 50 rpm. On day 1, 3, 5, 7, 10, 14 and 22, the PBS containing released BMP-2 was collected and replaced with fresh 2 ml of PBS. The collected samples were stored at −20°C before ELISA was conducted for the quantification of released BMP-2 from the particles (n=3). Optical density of wells in 96 well plate was measured using Molecular Devices Spectramax190 plate reader. In order to find the total BMP-2 encapsulated, the particles at the end of 22^nd day, were transferred into 1% acetic acid and incubated on same conditions for 24 h, to dissolve those particles. ELISA was again conducted to quantify the BMP-2 remaining in the solution.