DNA fragments in circulation released from apoptotic and necrotic cells were regarded as a novel prognostic or predictive biomarker for clinical diagnosis in recent years. However, DNA concentration in plasma ranged between 1 and 10 ng ml-1, which needed a single-molecule technology to analyze the base pair and concentration of DNA fragments. In this study, a series of different lengths of DNA fragments were studied, which showed that a good linear relationship between the DNA concentration and the molar concentration. The results suggested fluorescence correlation spectroscopy could access the nanomolar concentration of DNA labelled by SYBR Green I. Moreover, the relationship between the length of DNA fragment and the diffusion coefficient of DNA was scaled with the standard samples. The results demonstrates fluorescence correlation spectroscopy is a highly sensitive method for DNA detection.
The phosphatase and tensin homolog on chromosome 10 (PTEN) is one of important tumor suppressor proteins in ovarian cancer via negatively regulating the phosphatidylinositol 3-kinase–AKT signaling pathway and controlling genomic stability. Recent studies showed the physiological function of PTEN was closely related with its subcellular compartments. But only a few technologies could quantitatively measure the concentration of PTEN at different subcellular compartments in living cells. In this study, we used fluorescence correlation spectroscopy to measure the concentrations and dynamics of EGFP-PTEN in ovarian cancer cells HO-8910. Our results showed the increasing concentration of PTEN in the cytoplasm had an opposite trends with the nucleus after the oxidative stress stimulation which was induced by H2O2. Furthermore, the altered diffusion of PTEN at different subcellular compartments also illustrated the PTEN was trafficked from the cytoplasm to nucleus.
In this study, HOSEpiC ovarian cell was cultured on hydrogel substrates with three different Young moduli of 3, 19 and 35 kPa. Atomic force microscopy was used to measure the elasticity of cells on three different stiffness substrates. Furthermore, the distribution of actin filaments in HOSEpiC cell was observed by confocal imaging. From the measurements of atomic force microscopy, we found that substrate stiffness would cause changes of cellular elasticity. The largest one was on the substrate of 35 kPa, followed by the 19 kPa and cells on 3 kPa was the smallest. Besides, from the confocal imaging, it could be observed that the distribution of actin filaments in the cells was different on the three substrates. All these results showed that the elasticity of the cells was lower on the substrates with smaller stiffness, which indicated that cells appeared softer when the stiffness of substrate decreased.
Fluorescence correlation spectroscopy (FCS) is a powerful technique that could provide high temporal resolution and detection for the diffusions of biomolecules at extremely low concentrations. The accuracy of this approach primarily depends on experimental condition requirements and the data analysis model. In this study, we have set up a confocal-based FCS system. And then we used a Rhodamine6G solution to calibrate the system and get the related parameters. An experimental measurement was carried out on one-component solution to evaluate the relationship between a certain number of molecules and concentrations. The results showed FCS system we built was stable and valid. Finally, a two-component solution experiment was carried out to show the importance of analysis model selection. It is a promising method for single molecular diffusion study in living cells.
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