I'm using an Amplex Red kit, the reagent changes color to pink almost immediately in my own Krebs-Ringer buffer but not in HBSS. Why is this?
The components of Krebs-Ringer buffer (salts) should not cause oxidation of the Amplex reagent (which, in the presence of peroxidase and H2O2 oxidizes to resorufin, which is pink in color and fluorescent). Try water alone (the water used to make the Krebs-Ringer buffer). Since Hank's Buffered Saline Solution is typically purchased rather than made in the lab, it likely would not have the same contaminant. Another option is to degas the buffer prior to use to removed dissolved oxygen radicals.
Can Amplex Red Assays be performed using cell lysates?
This is not recommended. The presence of endogenous proteases can complicate the assay by degrading the horseradish peroxidase (HRP). Endogenous peroxidases and antioxidants can modify the H2O2 required for the reaction, competing with HRP (and catalase) for the substrate.
The Amplex Red Assays are best performed with either purified enzymes or extracted H2O2 in a defined buffer system, extracellular solutions or body fluids (media, serum, etc.) that do not exhibit high levels of endogenous protease or oxidase activity and do not contain antioxidants.
Glutamate excitotoxicity is involved in cell death caused by tributyltin in cultured rat cortical neurons.
Authors:Nakatsu Y, Kotake Y, Komasaka K, Hakozaki H, Taguchi R, Kume T, Akaike A, Ohta S,
Journal:Toxicol Sci
PubMed ID:16207939
'Tributyltin, an endocrine-disrupting chemical, has been used as a heat stabilizer, agricultural pesticide, and component of antifouling paints. In this study, the neurotoxicity of tributyltin was investigated in cultured rat cortical neurons. Tributyltin caused marked time- and dose-dependent increases in the number of trypan blue-stained cells. Measurement of extracellular glutamate ... More
Iron alters glutamate secretion by regulating cytosolic aconitase activity.
Authors:McGahan MC, Harned J, Mukunnemkeril M, Goralska M, Fleisher L, Ferrell JB
Journal:Am J Physiol Cell Physiol
PubMed ID:15613494
'Glutamate has many important physiological functions, including its role as a neurotransmitter in the retina and the central nervous system. We have made the novel observations that retinal pigment epithelial cells underlying and intimately interacting with the retina secrete glutamate and that this secretion is significantly affected by iron. In ... More
Bispecific small molecule-antibody conjugate targeting prostate cancer.
'Bispecific antibodies, which simultaneously target CD3 on T cells and tumor-associated antigens to recruit cytotoxic T cells to cancer cells, are a promising new approach to the treatment of hormone-refractory prostate cancer. Here we report a site-specific, semisynthetic method for the production of bispecific antibody-like therapeutics in which a derivative ... More
Single cell glutamate analysis in Aplysia sensory neurons.
Authors:Drake TJ, Jezzini S, Lovell P, Moroz LL, Tan W
Journal:J Neurosci Methods
PubMed ID:15848241
'Glutamate is the major excitatory neurotransmitter in the central nervous system. However, techniques and assays available for the determination and detection of glutamate are limited. Here we have applied an effective glutamate assay toward the high-throughput analysis of single neurons. Initial physiological studies and recent immunohistochemical data strongly suggested that ... More
P2X agonist BzATP interferes with amplex-red-coupled fluorescence assays.
Authors:León D, Marín-García P, Sánchez-Nogueiro J, de la O FO, García-Carmona F, Miras-Portugal MT,