By Gerald Thiel
This quantity presents present tips on how to study the homes of dressmaker receptors solely activated via clothier medications (DREADDs) in vitro and to degree the organic responses of DREADD activation in several neuronal populations in vivo. Chapters concentrate on the application of DREADD expertise to selectively stimulate or inhibit neuronal job in chosen mind components of transgenic mice. moreover, the profitable use of DREADD expression in rats and Drosophila is defined. jointly, the DREADD know-how represents a non-invasive, chemical-genetic software for investigating neuronal signaling and the ensuing behavioural responses in vivo. Written within the renowned Neuromethods series kind, chapters comprise the type of aspect and key recommendation from the experts had to get profitable leads to your individual laboratory. Concise and easy-to-use, Designer Receptors completely Activated through fashion designer medications aims to make sure winning ends up in the extra examine of this important field.
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Additional info for Designer Receptors Exclusively Activated by Designer Drugs
The cells are then incubated for 3–6 h at 37 °C, followed by the addition of 5 ml DMEM containing 20 % FBS. After this step, the cells are cultured for ~48 h and then used for various assays (see below). 3 Notes Cell density can greatly affect transfection efficiency. Cells that are 100 % confluent or less than 50 % confluent are usually not suitable for transfection. , pcDNA) should always be included in the experimental setup. 3 Receptor Binding Assays Radioligand binding experiments can be used to determine the presence, density, and pharmacological characteristics of DREADDs (or GPCRs in general) expressed in transfected cells.
Manassas, VA). The BCA Protein Assay Kit is from Thermo Scientific/Pierce (Rockford, IL). 2 Methods All buffers are kept cold (4 °C). Crude membranes are prepared from receptor-expressing COS-7 cells ~48 h after transfection. DMEM is removed via aspiration from the 100 mm plates containing the transfected cells. 4) is added to each dish, followed by a 15-min incubation on ice. 5 ml Eppendorf tube. This procedure is repeated once with 350 μl of binding buffer in order to collect the remaining cells.
On the next day, the vials are briefly vortexed and then transferred to a scintillation counter (count each vial for several minutes). Binding data are fitted to a one-site binding hyperbola via nonlinear regression analysis by using GraphPad Prism software. The amount of specific binding (total minus nonspecific binding) is plotted on the y-axis as a function of the [3H]-NMS concentration plotted on the x-axis. Specific binding is usually expressed as picomoles or femtomoles per mg protein. The extrapolated amount of [3H]-NMS bound by the receptor/DREADD when binding is saturated is defined as the number of receptor sites (Bmax).