No abstract

Intracellular free H + concentration (pHi) responds to numerous extracellular stimuli. The use of fluorescent indicator dyes to measure pHi is strongly influenced by the ability of target cells to retain activated dye within the cytoplasmic compartment. Here, 3 pHsensitive indicator dyes-acetoxymethyl (AM) esters of SNARF-1 and BCECF, and the thiol-reactive 5-chloromethyfluorescein (CMFDA)-were examined for monitoring pHi. The stability of pH measurements was strongly affected by temperature, cell type, indicator dye, and use of transport inhibitors to prevent dye export. Cellular retention of CMFDA, which forms covalent complexes, was sufficient to permit monitoring of transient pHi changes over extended time periods in a multi-well plate assay format. In human embryonic kidney (HEK293) and Chinese hamster ovary (CHO) cells, increasing osmotic pressure caused a significant rise in pHi. In contrast, activation of native or transfectedadrenergic, cholinergic, and d and m opioid receptors did not measurably affect pHi in HEK293 cells. Decreases in pHi were observed in CHO cells expressing the human H + /peptide transporter PEPT1 upon addition of dipeptide substrates. The use of CMFDA in multi-well formats should facilitate study of osmotic and transport activity and screening for drugs that affect pHi. laboratory described a rapid and sensitive assay to measure intracellular calcium from multi-well plates using a fluorometer equipped with on-line injectors. Similarly, Grant and Acosta 2 have described a ratiometric assay of pHi, using BCECF fluorescent dye and a multi-well plate reader. However, their assay was performed at room temperature rather than 37°C, required a ratiometric approach with 2 emission wavelengths measured, and did not permit rapid analysis of pH changes in the millisecond range.The purpose of this study was to develop a rapid fluorimetric assay using the multi-well plate format to measure intracellular pH change in live cells and examine pHi changes induced by various effectors. The principal advantages of using pH-sensitive dyes over other methods (eg, distribution of weak acids or bases, 31 P-NMR spectroscopy, ion-sensitive microelectrodes, pHsensitive Green fluorescent protein [GFP] mutants) are sensitivity, applicability to many cell lines, and continuous monitoring of rapid kinetic pH changes. However, the main disadvantage is often substantial dye leakage from the cells, especially at 37°C. Since we have observed that intracellular calcium responses can dramatically differ at 20°C and 37°C (unpublished data), one aim of this study was to find a dye providing a stable signal to allow pHi measurements at physiological temperature in several different cell lines. Furthermore, use of a plate reader with dual injectors (control and experimental samples) and the capability to take measurements in the millisecond scale would further enhance the utility of the assay. Last, this approach also enhances the accuracy of single-wavelength measurements, thereby reducing the time required for ...

In this work, a convenient test of antioxidant activity was developed, with BChE-contaminated HSA as the target of AAPH-induced oxidation and its esterase activity as the marker of protein integrity or degradation. The method is relatively simple, of low cost, and convenient to use. Its application to natural polyphenols showed that quercetin (1), verbascoside (2), chlorogenic acid (3), caffeic acid (4), 1,3,6,7-tetrahydroxyxanthone (5), and mangiferin (6), are good antioxidants (IC 50 < 9 mm). 1,5-Dihydroxy-3-methoxyxanthone (7), flemichin D (8), and cordigone (9) showed modest activities (ca. 50 mm < IC 50 < 350 mm), whereas danthrone (10) was inactive. Complementary experiments with two of the more active antioxidants, namely quercetin (1) and chlorogenic acid (3) showed that both antioxidants were better radical scavengers than chain-breaking antioxidants. The relative adiabatic oxidation potential (DH ox ), the relative H-bond dissociation energy (DH abs ), and the first oxidation potential measured by cyclic voltammetry were found to be related to the radical-scavenging activity of these antioxidants.