Cell Navigator® Live Cell Endoplasmic Reticulum (ER) Staining Kit *Red Fluorescence*

1. Prepare cells in growth medium
2. Incubate cells with ER Tracer™ Red working solution at 37 °C for 15 - 30 minutes
3. Analyze under fluorescence microscope at Ex/Em = 590/620 nm (TRITC or Cy3 filter set)

Important     Thaw all the kit components at room temperature before starting the experiment.

For guidelines on cell sample preparation, please visit https://www.aatbio.com/resources/guides/cell-sample-preparation.html

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles

Add 20 µL of DMSO (Component C) into the vial of ER Tracer™ Red (Component A) and mix well to make 500X ER Tracer™ Red stock solution.

Note        20 µL of 500X ER Tracer™ Red stock solution is enough for one 96-well plate. Unused 500X ER Tracer™ Red stock solution can be stored at ≤ -20 ºC for two weeks if the tubes are sealed tightly. Protect from light.

Add 20 µL of 500X ER Tracer™ Red stock solution into 10 mL of Live Cell Staining Buffer (Component B), and mix well to make ER Tracer™ Red working solution.

Note        This ER Tracer™ Red working solution is stable for at least 2 hours at room temperature. Protect from light.

1. Plate and treat cells as desired.
2. Remove cell culture medium. Optional: Cells can be washed twice with buffer of your choice.

3. Add 100 µL/well (96-well plate) or 50 µL/well (384-well plate) of ER Tracer™ Red working solution in the cell plate. Incubate cells with working solution at 37 °C for 15 - 30 minutes, protected from light.

   Note        The optimal concentration of the ER probe varies depending on the specific application. Concentration higher than the working solution can be toxic to cells. The staining conditions may be modified according to the particular cell type and the permeability of the cells or tissues to the probe.

4. Remove ER Tracer™ Red working solution in each well. Wash cells with physically relevant buffer three times.

5. Fix cells after staining (Optional). Fix the cells with 4% formaldehyde for 5 - 10 minutes. Wash cells with physically relevant buffer three times.

6. Observe the fluorescence signal in cells using fluorescence microscope with TRITC or Cy3 filter set (Ex/Em = 590/620 nm).