Transfection of Calcium-Sensing Receptor Mutants in HEK293 Cells

Human embryonic kidney (HEK) 293 cells were cultured in high-glucose Dulbecco’s modified Eagle medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum, as described (17 (link)). WT (Leu723) and mutant (Gln723) CaSR-pEGFP-N1 constructs were generated, as reported (16 (link)), and transiently transfected into HEK293 cells using Lipofectamine Plus (Invitrogen), as described (16 (link)). Successful transfection of WT and mutant CaSR proteins was confirmed by visualizing green fluorescent protein (GFP) fluorescence using an Eclipse E400 fluorescence microscope with an epifluorescence filter, and images were captured using a DXM1200C digital camera and NIS Elements software (Nikon, Tokyo, Japan), as described (17 (link)).

Free full text: Click here

Babinsky V.N., Hannan F.M., Ramracheya R.D., Zhang Q., Nesbit M.A., Hugill A., Bentley L., Hough T.A., Joynson E., Stewart M., Aggarwal A., Prinz-Wohlgenannt M., Gorvin C.M., Kallay E., Wells S., Cox R.D., Richards D., Rorsman P, & Thakker R.V. (2017). Mutant Mice With Calcium-Sensing Receptor Activation Have Hyperglycemia That Is Rectified by Calcilytic Therapy. Endocrinology, 158(8), 2486-2502.

Publication 2017

Dulbecco’s modified Eagle medium Lipofectamine Plus Eclipse E400 fluorescence microscope DXM1200C digital camera NIS Elements software

Camera Cells Cultured medium Eagle Embryonic Fetal bovine serum Fluorescence Fluorescence microscope Glucose Green fluorescent protein Hek293 cells Human Kidney Lipofectamine Mutant proteins Transfection

Corresponding Organization :

Other organizations : University of Oxford, Oxford Centre for Diabetes, Endocrinology and Metabolism, University of Liverpool, University of Ulster, Mary Lyon Centre at MRC Harwell, Medical Research Council, Medical University of Vienna, GlaxoSmithKline (United Kingdom)

Top 5 similar protocols

Protocol cited in 1 other protocol

Variable analysis

independent variables

WT (Leu723) and mutant (Gln723) CaSR-pEGFP-N1 constructs

dependent variables

Successful transfection of WT and mutant CaSR proteins
Green fluorescent protein (GFP) fluorescence

control variables

High-glucose Dulbecco's modified Eagle medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum

controls

Positive control: WT (Leu723) CaSR-pEGFP-N1 construct
Negative control: Not explicitly mentioned

Annotations

Based on most similar protocols

Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.

As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!