BrightER - Endoplasmic reticulum probe for live imaging & flow cytometry

This compound's ability to specifically target the ER was a serendipitous discovery, and we therefore do not have precise information on what the exact mechanism is, or which ER targets the probe is binding to. What we know from its chemical structure is that it is most likely targeting a protein, rather than a lipidic compound, therefore minimizing the risk for non-specific binding to other lipidic components of cellular membranes as seen with many commercial ER probes. 

BrightER specificity has been checked by co-localisation studies, showing strong correlation between BrightER and Transferrin Receptor (TfR) signals. Check our Results section for more details. 

BrightER can be used for live ER imaging. We recommend using a confocal microscope with high magnification instead of epifluorescence microscopy for optimal performance. 

Alternatively, BrightER can also be used for flow cytometry. A proportional increase in BrightER signal was found upon treatment with Thaps, demonstrating the potential of BrightER to serve as a simple and reliable ER stress marker. Another group successfully used BrightER in flow cytometry to investigate the role of an ER-resident protein, by measuring changes in signal upon inducible gene knock-down.  Check out our Results section for more details.   

BrightER signal has been shown to be highly stable over 3 hours once added to the cells, without any sign of cell toxicity or alteration of ER physiology.

Given the minimization of effects on ER physiology is of great importance, the BrightER's inventors performed a functional assay looking at intracellular protein trafficking following BrightER labeling. This assay showed that BrightER did not affect protein trafficking from the ER to the plasmic membrane. Check out our Results section for more details. 

No, BrightER can only be used on live samples and will not be retained after aldehyde fixation.  

One tube contains 100µL of dye, that we recommend diluting at a final concentration of 1:100 in culture medium. Therefore, you can make up to 10mL of final imaging medium with one vial. Optimal working concentration can be adjusted depending on cell type characteristics and applications (some users got a satisfactory fluorescence signal with a concentration down to 1:1000). 

BrightER is conjugated to a tetramethylrhodamine dye, with a peak excitation at 557 nm and peak emission at 576 nm. 

Aggregates can form in the BrightER solution. As stated in the protocol of use, performing two cycles of vortex (30sec) + heating at 70°C (90sec) should dissolve most granules. If remaining granules still cause an issue, you can wash out the imaging medium and replace with fresh medium without dye (2-3 washings might be necessary). Alternatively, sonicating and/or using Pluronic F-127 can help reduce BrightER aggregate formation. 

We do not recommend spinning down the tube and taking out the supernatant to get rid of aggregates, as this will lead to reduced signal intensity.

Given the probe is coupled to a strong rhodamine dye, its presence in the imaging medium indeed produces a background of fluorescence that can be problematic when imaging in epifluorescence microscopy. Potential ways to minimize imaging medium autofluorescence include:

• Using a phenol-free medium (i.e. FluoroBrite DMEM, ThermoFisher cat n.A1896701)
• After the 15 min incubation with BrightER, wash out the imaging medium  and replace with fresh medium without dye before imaging
• Control excitation intensity: adjust it to the minimum required for imaging
• Alternatively, when possible, using a confocal microscope should allow you to discriminate the BrightER signal present in cells from the background fluorescence signal, and therefore get a reliable ER signal in your cells

Although the recommended protocol of use has been shown to work with most cell types, optimal experimental conditions to use for ER labeling can vary depending on specific cell type characteristics and experimental goals. Several parameters (imaging buffer, incubation duration, dye concentration, etc) can be tuned to achieve optimal labeling. So far, the following variations have been used:

• Imaging buffer: cell culture medium, PBS & HBSS
• Incubation duration: up to 3 hours
• Dye concentration: 1:1000 to 1:100