Alkaline phosphatase activity

The alkaline phosphatase (ALP) activity of cells was examined after He-APPJ treatment. MC3T3-E1 osteoblasts (1×10⁴) were seeded in plates. After the cells became confluent, they were treated with plasma for 0, 1, 3, or 5 minutes. Then, the cells were incubated at 37°C in 5% CO₂ for 7 days, and ALP activity was measured in accordance with the manufacturer's instructions (Anaspec Co., Fremont, CA, USA). The cell lysates and ALP substrates were transferred to plates and incubated at 37°C for 30 minutes. The reaction was terminated by adding a stop solution. The p-nitrophenol (pNP) product formed by enzymatic hydrolysis of p-nitrophenylphosphate was measured at 405 nm using an absorbance microplate reader. The protein concentrations in the samples were measured using a protein assay kit (iNtRON Biotechnology, Seongnam, Korea). ALP activity was expressed as the concentration of pNP transformed per microgram of protein [14].