Elisa assay kit

Human VEGF-A was quantified in the cell culture supernatant using ELISA assay kit from R&D Systems, Inc. (Minneapolis, MN) as described previously [23 (link)]. Briefly, ECs were transfected with siRNA or transduced with adenovirus. After 24 hours, cells were washed with phosphate-buffered saline (PBS) and fresh medium was added. Supernatants of the cells were collected at 48 hours and the number of cells was counted. An ELISA assay was performed according to the manufacturer's protocol. After measuring the VEGF-A content in the supernatants, the amount of VEGF-A per 10⁶ cells was calculated. Each experiment was repeated three independent times.

Frozen tissues were homogenized, then a BCA Protein Assay Kit (Solarbio, Beijing, China) was used for the quantitative determination of total protein concentration in the supernatants. Plasma and tissue supernatants were diluted to suitable concentrations and measured using an ELISA assay kit (R&D Systems, Minneapolis, MN, USA).

Cytokines in BALF and plasma, including interferon-γ (IFN-γ), tumor necrosis factor (TNF-α), interleukin-1 beta (IL-1β), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-12 (IL-12p70), and transforming growth factor beta (TGF-β1) were measured with an ELISA assay kit (R&D Systems, MN, USA). The z-score transformation was utilized to calculate the relative level of cytokines according the following equation: Z= $\frac{X-\overline{X}}{\mathit{SD}}$ ( $\overline{X}$ is the mean value, SD is standard deviation).

The RAW264.7 cells were incubated on the bone cements for 1 and 3 days. The cell supernatant was used to the measure cytokine concentration using an ELISA assay kit (R&D Systems, United States). The operation was done according to the manufacturer’s instructions and calibrated by standard curves. Three samples were tested in each group.

Plasma was isolated during PBMC separation and supernatants were collected from confluent cell line cultures. They were maintained in −80 °C. The samples were resolved at the same time, and the concentrations of M-CSF and VEGF in these samples were measured using ELISA assay kit (R&D, Minneapolis, MN) following the manufacturer’s instructions.

The SOD, MDA, CAT, T-AOC, and TBARS contents in the liver tissue were measured using commercially available assay kits. The levels of AST and ALT in serum were assessed by an ELISA assay kit (RD system) according to the manufacturer’s instructions. The levels of AGEs in the serum were determined using a commercial reagent kit. All biochemical analyses were performed according to the manufacturer's protocols.

20 mg growth factor loaded microspheres were immersed in 2 ml PBS and then oscillated in 37°C constant temperature water bath for 28 days. 1 ml supernatant was aspirated and replaced with fresh phosphate buffer saline (PBS, pH 7.4, Hyclone) every 24 h. The amount of released BMP-2 or VEGF in the supernatant was detected, respectively, using ELISA assay kit (R&D) according to the instructions. After being washed, diluted standards or samples were added into each well, followed by being incubated with diluted detected antibody at room temperature for 2 hours. Then wells were washed again 6 times and filled with diluted Streptavidin-HRP to incubate at room temperature for 45 minutes, followed by being incubated with substrate solution away from light for 30 minutes at room temperature. After adding stop solution, the optical density of wells can be determined using microplate reader under double wavelength of 450 and 570/ 630 nm within 30 minutes. Concentration of samples can be calculated according to a formula derived from concentration and optical density of standards, then accumulative release curves were plotted to describe the release behavior of BMP-2/VEGF.