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.
Use of colorimetric test strips for monitoring the effect of hemodialysis on salivary nitrite and uric acid in patients with end-stage renal disease: a proof of principle.
Authors:Blicharz TM, Rissin DM, Bowden M, Hayman RB, DiCesare C, Bhatia JS, Grand-Pierre N, Siqueira WL, Helmerhorst EJ, Loscalzo J, Oppenheim FG, Walt DR,
Journal:Clin Chem
PubMed ID:18676588
'BACKGROUND: Initial screening of potential biomarkers for monitoring dialysis was performed with saliva samples collected from patients with end-stage renal disease (ESRD). A more thorough analysis of the most promising markers identified in the initial screening was conducted with saliva samples acquired at hourly intervals throughout dialysis to monitor analyte ... More
HucR, a novel uric acid-responsive member of the MarR family of transcriptional regulators from Deinococcus radiodurans.
Authors:Wilkinson SP, Grove A,
Journal:J Biol Chem
PubMed ID:15448166
'The MarR family of transcriptional regulators comprises a subset of winged helix DNA-binding proteins and includes numerous members that function in environmental surveillance of aromatic compounds. We describe the characterization of HucR, a novel MarR homolog from Deinococcus radiodurans that demonstrates phenolic sensing capabilities. HucR binds as a homodimer to ... More
Fructose induces transketolase flux to promote pancreatic cancer growth.
'Carbohydrate metabolism via glycolysis and the tricarboxylic acid cycle is pivotal for cancer growth, and increased refined carbohydrate consumption adversely affects cancer survival. Traditionally, glucose and fructose have been considered as interchangeable monosaccharide substrates that are similarly metabolized, and little attention has been given to sugars other than glucose. However, ... More
Activation of NLRP3 inflammasome in alveolar macrophages contributes to mechanical stretch-induced lung inflammation and injury.
Authors:Wu J, Yan Z, Schwartz DE, Yu J, Malik AB, Hu G,
Journal:J Immunol
PubMed ID:23436933
Mechanical ventilation of lungs is capable of activating the innate immune system and inducing sterile inflammatory response. The proinflammatory cytokine IL-1ß is among the definitive markers for accurately identifying ventilator-induced lung inflammation. However, mechanisms of IL-1ß release during mechanical ventilation are unknown. In this study, we show that cyclic stretch ... More
IL-1R1/MyD88 signaling is critical for elastase-induced lung inflammation and emphysema.
Authors:Couillin I, Vasseur V, Charron S, Gasse P, Tavernier M, Guillet J, Lagente V, Fick L, Jacobs M, Coelho FR, Moser R, Ryffel B,
Journal:J Immunol
PubMed ID:20007584
Lung emphysema and fibrosis are severe complications of chronic obstructive pulmonary disease, and uncontrolled protease activation may be involved in the pathogenesis. Using experimental elastase-induced acute inflammation, we demonstrate here that inflammation and development of emphysema is IL-1R1 and Toll/IL-1R signal transduction adaptor MyD88 dependent; however, TLR recognition is dispensable ... More