Opsys mr 96 well microplate reader

For the determination of Il-6 secretion, the medium of BMDMs from littermate wildtype (Cdk5^flox) and Cdk5^LysMCre mice after 4 h treatment with PBS as control, LPS (100 ng/ml), Dex (10⁻⁶ M) or LPS + Dex (100 ng/ml LPS and 10⁻⁶ M Dex) was collected, sterile filtered (0.2 μm) and stored at −80°C until measurement was performed. The Il-6 ELISA was performed with the Mouse Il-6 ELISA set (BD OptEIA^™) according to the manufacturer's protocol. The absorption was measured using the Dynex Opsys MR 96-Well Microplate Reader at 405 nm with a correction wavelength of 650 nm.

Calcium deposition on the HOB seeded 3D printed scaffolds was determined by Alizarin red S staining of HOB cells cultured on the scaffolds at day 21. On the day of measurement, the scaffolds were washed with PBS, fixed with 10% formal saline, and then washed with deionised water. The fixed specimens were stained with 1 mL 2% Alizarin red S for 15 min and then washed with 50% ethanol. Images of the stained scaffolds were taken for qualitative assessment. Quantitative assessments were conducted by adapting a previously published protocol [49 (link)]. The bound stain on the scaffolds was dissolved with 1 mL of 10% cetylpyridium chloride (CPC) solution. Aliquots (100 μL) of the supernatant were read using a microplate reader (Opsys MR™ 96-well microplate reader, Dynex Technologies, Chantilly, VA, USA) at 570 nm. Serial dilutions of known concentrations of Alizarin red S in CPC solution were read under the microplate reader to obtain the standard curve of Alizarin red S. Cell mineralization results were expressed as μ moles of calcium per well as 1 mole of Alizarin red S binds to 2 moles of calcium in an Alizarin red S-calcium complex [50 (link)]. Measurements from the blank control (unseeded scaffolds) were subtracted from the calcium deposition measurements of seeded scaffolds.