I have conducted a study on UV-induced nitric oxide production on human keratinocyte cell lines (HaCaT). The sources of the cells I used were from CLS and ATCC. I cultured the cells using general method until they were ready for the experiments. Unfortunately, the cells from both sources did not produce nitric oxide after UVB irradiation. In addition, I used Griess reagent system for the detection of nitric oxide in culture media. Nitric oxide in culture media was spectrophotometrically estimated at 24, 48 and 96 h after UV-irridiation. There was no nitric oxide in both control group (no UV) and experimental group. Nevertheless, I found that many groups of researcher reported that nitric oxide was released from keratinocyte and HaCaT cells after UVB irradiation. I followed the method they used. I varied the UV-dose, exposure area, and also the amount of cells. Still, I cannot detect any change in nitric oxide production.
Are there anyone use to conduct the similar experiment?
Am I miss any important thing in my experiment?
Is keratinocyte produce nitric oxide in response to UV-irradiation?