ER-Tracker Green (ERTG) is a cell permeable live cell stain for live cell imaging.ER-Tracker Green has an excitation wavelength of 504 nm and an emission wavelength of 511 nm.

The cellular autophagy of mitochondria and endoplasmic reticulum (ER) is referred to as mitophagy and ER-phagy, respectively, and is assessed using organelle quality probes MitoTracker Green (MTG) and ER Tracker Green (ERTG) in flow cytometry assays[4].

Instructions
I. Solution preparation
1. Stock solution: Dissolve ER-Tracker Green (ERTG) in DMSO to prepare a stock solution with a concentration of 10 mM. Avoid strong light exposure when preparing the solution because ERTG is light-sensitive.
2. Working solution: Dilute the stock solution to the desired concentration, usually 100nm–5 µM, using appropriate culture medium (such as DMEM, RPMI) or serum-free culture medium.
II. Application steps:
Live cell staining
1. Culture cells in appropriate culture medium until the desired confluence is reached.
2. Add working solution to live cells and incubate at 37°C for 30–60 minutes.
3. During incubation, ensure that cells are protected from light to avoid dye degradation.
4. After incubation, gently wash cells with culture medium or PBS to remove excess dye.
5. Imaging: Observe the stained cells using a fluorescence microscope. ERTG is excited at 504 nm and emits fluorescence at 511 nm.
Notes: For optimal imaging, use filters suitable for green fluorescence.
6. Calibration and Controls:
1) Control group: Set up a cell group without ER-Tracker Green to ensure specificity of staining.
2) Standard curve: If necessary, cells with known concentrations of ER-Tracker Green can be stained to establish a standard curve between fluorescence intensity and dye concentration.
Notes:
1) Photosensitivity: ER-Tracker Green is light sensitive. Avoid exposing the dye and stained cells to strong light during the experiment, especially during staining and imaging.
2) Storage conditions: ER-Tracker Green stock solution should be stored at -20°C in the dark to prevent degradation. Avoid repeated freezing and thawing.
3) Cell viability: Although ERTG is designed for live cell imaging, cell viability should be monitored because long-term exposure to high concentrations of dye may affect cell function.