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.
Phospholipase D1 production of phosphatidic acid at the plasma membrane promotes exocytosis of large dense-core granules at a late stage.
Authors:Zeniou-Meyer M, Zabari N, Ashery U, Chasserot-Golaz S, Haeberlé AM, Demais V, Bailly Y, Gottfried I, Nakanishi H, Neiman AM, Du G, Frohman MA, Bader MF, Vitale N
Journal:J Biol Chem
PubMed ID:17540765
'Substantial efforts have recently been made to demonstrate the importance of lipids and lipid-modifying enzymes in various membrane trafficking processes, including calcium-regulated exocytosis of hormones and neurotransmitters. Among bioactive lipids, phosphatidic acid (PA) is an attractive candidate to promote membrane fusion through its ability to change membrane topology. To date, ... More
Cholesterol distribution in the Golgi complex of DITNC1 astrocytes is differentially altered by fresh and aged amyloid beta-peptide-(1-42).
Authors:Igbavboa U, Pidcock JM, Johnson LN, Malo TM, Studniski AE, Yu S, Sun GY, Wood WG
Journal:J Biol Chem
PubMed ID:12584199
'The Golgi complex plays an important role in cholesterol trafficking in cells, and amyloid beta-peptides (Abetas) alter cholesterol trafficking. The hypothesis was tested that fresh and aged Abeta-(1-42) would differentially modify Golgi cholesterol content in DINTC1 astrocytes and that the effects of Abeta-(1-42) would be associated with the region of ... More
The antisignaling agent SC-alpha alpha delta 9, 4-(benzyl-(2-[(2,5-diphenyloxazole-4-carbonyl)amino]ethyl)carbamoyl)- 2-decanoylaminobutyric acid, is a structurally unique phospholipid analogue with phospholipase C inhibitory activity.
Authors:Vogt A, Pestell KE, Day BW, Lazo JS, Wipf P,
Journal:Mol Cancer Ther
PubMed ID:12481409
'Phospholipids and lipid second messengers mediate mitogenic signal transduction and oncogenesis, but there have been few successful examples of small molecules that affect biologically important phospholipid metabolism. Here we investigated the actions of a previously described antitumor agent, 4-(benzyl-(2-[(2,5-diphenyloxazole-4-carbonyl)amino]ethyl)carbamoyl)- 2-decanoylaminobutyric acid (SC-alpha alpha delta 9), which has antisignaling properties, on ... More
Interfacial sensing by alveolar type II cells: a new concept in lung physiology?
Authors:Ravasio A, Hobi N, Bertocchi C, Jesacher A, Dietl P, Haller T,
Journal:Am J Physiol Cell Physiol
PubMed ID:21270294
'Alveolar type II (AT II) cells are in close contact with an air-liquid interface (I(AL)). This contact may be of considerable physiological relevance; however, no data exist to provide a satisfying description of this specific microenvironment. This is mainly due to the experimental difficulty to manipulate and analyze cell-air contacts ... More
Loss of the ceramide transfer protein augments EGF receptor signaling in breast cancer.
Authors:Heering J, Weis N, Holeiter M, Neugart F, Staebler A, Fehm TN, Bischoff A, Schiller J, Duss S, Schmid S, Korte T, Herrmann A, Olayioye MA,
Journal:Cancer Res
PubMed ID:22472120
'Triple-negative breast cancers (TNBC) are especially refractory to treatment due to their negative hormone receptor and ErbB2/HER2 status. Therefore, the identification of cancer-associated deregulated signaling pathways is necessary to develop improved targeted therapies. Here, we show that expression of the ceramide transfer protein CERT is reduced in TNBCs. CERT transfers ... More