Jinfang Liao

A Novel Fluorogenic Coumarin Substrate for Monitoring Acid Phosphatase Activity at Low pH Environment

This article described the synthesis and application of 6-chloro-8-fluoro-4-methylumbelliferone phosphate (CF-MUP) in analyzing acid phosphatase activity. Compared to the existing MUP, the new coumarin phosphate, CF-MUP, demonstrateed much higher sensitivity and was more robust for detecting the activity of acid phosphatase than the classic substrate 4-methylumbelliferone phosphate (MUP). The product of enzyme reaction, 6-chloro-8-fluoro-4-methylumbelliferone (CF-MU) possesses strong fluorescence at ~450 nm with low pKa (4.7), high fluorescence quantum yield and pH independence in the physiological pH range. This new fluorescence dye, CF-MU, is a convenient tool for assays with buffer pH between 4.5 and 8.

Fluorescent real-time quantitative measurements of intracellular peroxynitrite generation and inhibition

Peroxynitrite (ONOO-), a strong oxidant species, is produced by the reaction of nitric oxide (NO) and superoxide (O2.-) radicals. It plays an important role as a biological regulator in numbers of physiological and pathological processes. In this study, we developed fluorescence-based real-time quantitative measurements to detect intracellular ONOO-. The probe DAX-J2 PON Green showed high selectivity toward ONOO- over other competing species, and has been successfully applied in microplate reader and flow cytometer to quantitatively measure endogenous ONOO- production. Moreover, the results demonstrated the inhibitory effects of curcumin on intracellular ONOO- generation.

Functional analysis of G-protein coupled receptors using a new fluorescein lactone-based intracellular calcium indicator

Calcium flux assays are preferred methods in drug discovery for characterizing G-protein coupled receptors (GPCRs) and screening GPCR agonists and antagonists. A new fluorescent Ca2+ indicator has been developed to provide improved fluorescence-based assays for detecting intracellular Ca2+ mobilization of GPCR targets. This first fluorescein lactone-based Ca2+ indicator has >100 times fluorescence enhancement upon calcium binding. It has an intracellular Ca2+-induced fluorescence signal more than ∼2 fold brighter than Fluo-4 AM, and ∼4 times brighter than Fluo-3 AM, the two most popular fluorescent Ca2+ indicators.

A Non-Radioactive Enzymatic Photometric Assay for Glucose Uptake in Insulin-Responsive 3T3-L1 Adipocytes

Glucose transport systems are responsible for transporting glucose across cell surface membranes. The rate of glucose uptake in cells is tightly regulated by factors including insulin. For example, increased glucose uptake may relate to high rate of proliferation in cancer cells. Screening for glucose uptake inhibitor is a new way to search for cancer treatments. Monitoring uptake of glucose has been dominated by a traditional radioactive measurement, which is costly and time consuming, and requires a special handling procedure.

Functional Analysis of GPCR and Calcium Channel Targets Using Quest Fluo-8

Calcium flux assays are preferred methods in drug discovery for screening G protein coupled receptors (GPCRs) and calcium channels. Quest Fluo-8 AM, Fluo-3 AM and Fluo-4 AM are evaluated for several GPCR and ion channel targets. They share the same assay principle. All the three fluorogenic calcium indicators are in the form of non-fluorescent AM esters. Once inside cells, the lipophilic AM blocking groups are cleaved by non-specific cellular esterases, resulting in negatively charged fluorescent dyes that stay inside cells, and their fluorescence intensities are greatly enhanced upon binding to calcium. When cells stimulated with bioactive compounds, the receptor signals release of intracellular calcium, which greatly increase their fluorescence signals. In conclusion, Fluo-3 AM, Fluo-4 AM and Quest Fluo-8 AM are robust tools for evaluating GPCR and calcium channel targets and screening their agonists and antagonists with fluorescence microplate readers, fluorescence microscopes or flow cytometers.