Methods Characterisation of osteoblasts

2.2.1 Materials

The alkaline phosphatase assay, components of the Past Red dye, Triton X-100 and Bovine serum albumin (BSA) were obtained from Sigma-Aldrich Company Ltd (Poole, Dorset, UK). The osteocalcin assay was a Novocalcin assay obtained from Metra Biosystems via their UK distributor Quidel (Great Haseley, Oxon, UK).

2.2.2 Introduction

All cells used in this study were tested for osteoblast characteristics using the following techniques:

• Alkaline phosphatase biochemical assay;

• Alkaline phosphatase histochemical staining; and • Osteocalcin ELISA.

The alkaline phosphatase assay provides a measure of alkaline phosphatase enzyme activity. The assay uses p-nitrophenol phosphate as a substrate that is converted to p- nitrophenol and inorganic phosphate by alkaline phosphatase. Addition of alkali to the assay converts p-nitrophenol into a coloured product that can be measured by a spectrophotometer. The assay was carried out following the manufacturer’s protocol although the volume of reagents used was scaled down to a 96 well plate format. The osteocalcin assay is a competitive immunoassay. A mouse anti-human osteocalcin antibody binds to secreted osteocalcin present in culture medium. This is then detected by an anti-mouse alkaline phosphatase conjugate and a p-nitrophenol substrate, forming a coloured product that can be measured using a spectrophotometer.

2.2.3 Alkaline phosphatase assay

Cells at sub-confluency were lysed using 0.1% triton X-100, diluted in PBS. Lysates were stored at -20°C prior to use. On the day of the experiment, cell lysate was removed from the freezer and thawed at room temperature. The supernatant was then vortexed to mix, spun down briefly in a microfuge and then placed on ice. The BSA protein assay was then carried out to determine the amount of protein present in each sample of supernatant following the manufacturer’s instructions. The alkaline phosphatase assay was also carried out following the manufacturer’s instructions, outlined here:

• Alkaline phosphatase buffer solution and stock substrate solution were placed in a 37°C water bath for 15 minutes

• 5 ^il of 0.1% triton X-100 (diluted in PBS) was added to the triplicate of blanks • 5 pil of each cell lysate sample was added in triplicate

• The plate was incubated at 37°C for 15 minutes in an incubator • After the incubation, 50fxl of 0.05N NaOH was added to each well.

The absorbance was measured at 405nm using a spectrophotometer and the mean absorbance of the blanks was subtracted from each of the test absorbance readings.

In order to derive the standard curve, a 96 well plate was set up using the p- nitrophenol standard solution (diluted in 0.02N NaOH) to the following dilutions: 0.006 p,mol, 0.0125 pimol, 0.025pimol and 0.05p,mol. Each dilution was carried out in triplicate with a triplicate of NaOH used as a blank. The absorbance was measured at 405nm using a spectrophotometer and the mean absorbance of the blanks was subtracted from each of the readings. The equation of the standard curve was derived and used to determine the activity of alkaline phosphatase in each sample.

Each experiment was repeated three times on separate occasions and the mean enzyme activity value and standard deviation was obtained from the three repeats. This was then represented graphically.

2.2.4 Alkaline phosphatase histochemical staining

A stock solution of 0.2mg.ml'^ of napthol AS-MX phosphate was dissolved in 1ml N,N dimethylformamide (the dimethylformamide was diluted in O.IM Tris buffer pH9.2).

Immediately before use Img.ml'^ of Fast Red was added to the above stock solution. Medium was removed from the cells and then the cells were washed twice in PBS and once in 0.2mg.ml"^ napthol AS-MX phosphate stock solution. Fast Red solution was added to the cells and left to develop for 2-5 minutes at room temperature. Cells were then fixed with 4% paraformaldehyde.

2.2.5 Osteocalcin assay

Cell supernatants were removed from cells in culture and stored at -20°C prior to use. On the day of the experiment, cell supernatant was removed from the freezer and thawed at room temperature. The cell supernatant was then vortexed to mix and placed on ice. The assay was carried out following the manufacturer’s instructions:

The osteocalcin-coated strips were labelled and placed in the holder provided 25p.l of sample, osteocalcin standards or control (provided by manufacturer) was placed into the wells of the osteocalcin coated strips

125[xl of anti-osteocalcin antibody was added to each well and incubated at room temperature for two hours

After the incubation, the strips were inverted to empty and washed in 500p,l of Ix wash buffer (provided by the manufacturer)

This was repeated twice more and then the strips were then blotted to dry on paper towels

150p,l of enzyme conjugate was added to each well and incubated for 60 minutes at room temperature

The strips were washed a total of three times, as above, and blotted to dry on paper towels

150fxl of working substrate solution was added to each well and incubated for 40 minutes

After the incubation, 50|il of 3N NaOH was added to each well

The strip was then read at 405nm using a spectrophotometer and a standard curve was constructed using the absorbance readings from the known standards. The equation of this line was used to work out the concentration of osteocalcin present in the samples.