The endoplasmic reticulum (ER) structure is composed from the outer nuclear envelope, and consists of sheets and tubules. Here we describe a variety of labels available to distinguish morphology in both live and fixed cells. These ER labels are highly selective, allowing researchers to identify the ER and follow cellular behavior.
Endoplasmic reticulum introduction
The endoplasmic reticulum (ER) is found in all eukaryotic cells and is the largest organelle, consisting of more than half the total membrane content. Its structure is a continuation from the outer layers of the nuclear membrane, consisting of sac-like sheets called cisternae, and branched tubules [1]. The flattened sac-like sheet come in two forms: membrane bound by ribosomes (rough ER) or unbound by ribosomes (smooth ER). The ER has a central role in lipid and protein synthesis, protein chaperoning and folding, and calcium homeostasis [1].
Selection guide for endoplasmic reticulum stains
| ER tracker dyes | ER fluorescent fusion proteins | ||||
|---|---|---|---|---|---|
| ER-Tracker Blue-White DPX | ER-Tracker Green (BODIPY FL Glibenclamide) | ER-Tracker Red (BODIPY TR Glibenclamide) | CellLight ER-GFP, BacMam 2.0 | CellLight ER-RFP, BacMam 2.0 | |
| Readout | Live cell ER staining | Expression of fluorescent fusion protein | |||
| Target | Binds to K+ channels in ER | Protein retained in ER lumen | |||
| Common filter set | DAPI | FITC | Texas Red | FITC | TRITC |
| Labels | ER-Tracker Blue-White | BODIPY FL | BODIPY TR | GFP | RFP |
| Ex/Em (nm) | 374/430–640 | 504/511 | 587/615 | 488/520 | 555/584 |
| Signal-to-noise ratio |  | | |  | |
| Photostability | | | | | |
| Multiplexing | Yes | Yes | Yes | Yes | Yes |
| Live cells | Yes | Yes | Yes | Yes | Yes |
| Fixed cells | No | No | No | No | No |
| Fixable | Yes | Yes | Yes | Yes | Yes |
| Platform | Imaging | Imaging | Imaging | Imaging | Imaging |
| Format | 1 mM | 100 μg | 100 μg | 1 mL | 1 mL |
| Cat. No. | E12353 | E34251 | E34250 | C10590 | C10591 |
| For ER labeling of fixed cells, SelectFX Alexa Fluor 488 Endoplasmic Reticulum labeling kit provides all the reagents required to fix and permeabilize mammalian cells and then specifically label the ER. This kit contains an antibody directed against the ER-associated protein disulfide isomerase (PDI) and Alexa Fluor 488 dye-labeled secondary antibody. | |||||
Endoplasmic reticulum tracker dyes
ER-Tracker dyes are highly selective, cell-permeant, live cell endoplasmic reticulum stains. At low concentrations, these dyes have not been shown to be toxic to cells. When cells are stained using the protocol provided, the ER staining pattern is partially retained after fixation with formaldehyde.
ER-Tracker Blue-White DPX is highly selective and photostable. It has an excitation ~374 nm and is environment sensitive resulting in an emission range from 430 nm to 640 nm. While a standard DAPI filter works best and is recommended, a UV longpass filter can be used when visualizing ER staining.
ER-Tracker Green (Figure 1) and ER-Tracker Red (Figure 2) stains, BODIPY FL glibenclamide and BODIPY TR glibenclamide, respectively, are fluorescent sulfonylureas. These ER stains are targeted using glibenclamide, which binds to the sulfonylurea receptors of ATP-sensitive K+ channels that are prominent on the ER; however, they may have more disseminated tissue- and cell type-dependent distributions. It is important to note that the pharmacological activity of glibenclamide could potentially affect ER function.
Figure 1. Organelle labeling in live cells. Human osteosarcoma (U-2 OS) cells were labeled with CellMask Deep Red plasma membrane stain, the endoplasmic reticulum-selective ER-Tracker Green dye, mitochondrion-selective MitoTracker Red CMXRos dye, and blue fluorescent NucBlue Live Cell Stain. Labeled cells were imaged in the Live Cell Imaging Solution on the Zeiss 710 point-scanning spectral detection confocal microscope.
Figure 2. Organelle labeling in HeLa cells. HeLa cells labeled using NucBlue Live ReadyProbes Reagent, MitoTracker Green FM, ER-Tracker Red, and Tubulin Tracker Deep Red show multiplexing capability and staining specificity. Cells were imaged in Gibco HBSS buffer containing calcium and magnesium, supplemented with 1X Probenecid solution. Images were generated using an EVOS FL Auto 2 Imaging System with an Olympus 60X Super Apochromat Oil objective using DAPI, GFP, Texas Red, and Cy5 EVOS light cubes.
Endoplasmic reticulum fluorescent fusion proteins
CellLight fluorescent fusion proteins can be used for endoplasmic reticulum staining, allowing for identification and demarcation of the ER in live cells and to follow the dynamics of cellular behavior. CellLight ER-GFP (Figure 3) and CellLight ER-RFP (Figure 4) are ready-to-use constructs that express fluorescent proteins with ER-retention signals from calreticulin (1.8 kDa, N-term), a resident protein of the ER lumen, and KDEL (0.5 kDa, C-term). These fusion proteins are highly specific since localization is directed by cellular protein trafficking.
Introducing CellLight fluorescent fusion proteins involves a simple transfection step using the BacMam technology, and they work like cell stains with minimal toxicity or chemical disruption. These ER fusion proteins are compatible with other fluorescent probes for multiplex analysis in live cells, or after formaldehyde fixation for colocalization studies.
Learn more about these and other CellLight fluorescent proteins
Figure 3. Live cell imaging with CellLight ER-GFP. USOS cells transduced with CellLight ER-GFP. The following day, cells were co-stained with Hoechst 33342 and imaging was performed on live cells using a DeltaVision Core microscope and standard DAPI/FITC filter sets.
Figure 4. Live-cell imaging with CellLight ER-RFP and MitoTracker Deep Red FM. HeLa cells were transduced with CellLight ER-RFP and co-stained with MitoTracker Deep Red FM and Hoechst 33342. Imaging was performed on live cells using a Delta Vision Core microscope and standard DAPI/TRITC/Cy5 filter sets.
References
Resources
Molecular Probes Handbook
BioProbes 70 Journal article
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