1. Preparation of ER-Tracker Working Solution

1.1 Preparation of ER-Tracker Stock Solution The stock solution should be prepared in dimethyl sulfoxide (DMSO, concentration 1 mM. If the stock solution is to be applied to an unused solution, it is necessary to aliquot and refreeze it to a temperature of at least -20°C. It is important to avoid repeated freeze/thaw cycles. The solution can be stored for up to 6 months.)

1.2 Preparation of ER-Tracker working solution

The stock solution should be diluted with serum-free cell culture medium or PBS in order to obtain a working solution with a concentration ranging from 100nM to 1μM. It is recommended that the working solution be prepared in advance for use.

Please note that the final concentration of the working solution is dependent on the experimental system. It is therefore recommended that the final concentration conditions be optimised.

2. Cell Staining

2.1 Suspended Cells

2.1.1 Centrifuge at 1000 g for 3-5 minutes at 4°C. Then, discard the upper layer and wash twice with PBS for 5 minutes each time.

2.1.2. Please add 1 mL of working solution and incubate at room temperature for 5-30 minutes.

2.1.3 Centrifuge at 400g for 3-4 minutes at 4°C. Please dispose of the item immediately.

2.1.4. It is recommended that the item be washed with PBS on two separate occasions, each time for a duration of five minutes.

2.1.5. The cells should then be resuspended in serum-free cell culture medium or PBS. The observation of this process should be carried out using a fluorescence microscope or a flow cytometer.

2.2 Adherent cells

2.2.1. The cells which adhere to the culture should be placed on sterile coverslips.

2.2.2. The coverslip should then be removed from the medium and the excess medium aspirated.

2.2.3. Next, add 100 μL of working solution, shake gently to ensure full coverage of the cells, and incubate for 5-30 minutes at room temperature.

2.2.4. Please ensure that the product is washed twice with the culture medium, for a duration of five minutes on each occasion. The observation of this sample should be conducted using a fluorescence microscope or a flow cytometer.

Please note that if flow cytometry is to be used, the cells must be resuspended and then stained.