This plan only provides a guide, please modify it to meet your specific needs.
1. Preparation of staining solution
(1) Prepare DMSO storage solution: Dissolve ER-Tracker Green in DMSO and prepare a storage solution with a concentration of 1mM.
Note: Unused storage solution should be aliquoted and stored in the dark at -20°C or -80°C to avoid repeated freezing and thawing.
(2) Prepare working solution: Dilute the storage solution with a suitable buffer (such as serum-free medium or PBS) and prepare a working solution with a concentration of 100 nM-1 μM.
Note: Please adjust the concentration of the working fluid according to the actual situation and prepare it now.
2. Cell suspension staining
(1) Suspension cells: Centrifuge at 1000g for 3-5 minutes at 4°C, discard the supernatant, and wash twice with PBS for 5 minutes each time.
(2) Adherent cells: Wash twice with PBS, add trypsin to digest the cells, and centrifuge at 1000g for 3-5 minutes after digestion is completed.
(3) Add 1mL of DiOC6 (3) iodide working solution and resuspend about 106 cells. Incubate at room temperature in dark for 5-30 minutes. The optimal incubation time for different cells is different, please explore by yourself according to the specific experimental needs.
(4) After the incubation, centrifuge at 1000g for 5 minutes, remove the supernatant, add PBS and wash 2-3 times, 5 minutes each time.
(5) Resuspend the cells in pre-warmed serum-free cell culture medium or PBS. Observe by fluorescence microscopy or flow cytometry.
3. Cell adhesion staining
(1) Culture adherent cells on sterile coverslips.
(2) Remove the coverslip from the culture medium, suck out the excess culture medium, and place the coverslip in a humid environment.
(3) Add 100μL of dye working solution from one corner of the coverslip and shake gently to evenly cover all cells with the dye.
(4) Incubate at room temperature in the dark for 5-30 minutes. The optimal incubation time for different cells is different, please explore by yourself according to the specific experimental needs.
(5) After the incubation, discard the dye working solution and use pre-warmed culture solution to wash the coverslip 2 to 3 times.
4. Microscope detection: The maximum excitation/emission light of ER-Tracker Green is 489/520 nm respectively.
Precautions:
① If you want to fix the stained cells, it is recommended to use 4% formaldehyde for 2 minutes at 37℃;
② If the stained cells need to be permeabilized, it is recommended to use ER–Tracker Blue–White DPX (GB30171);
③ Fluorescent dyes all have quenching problems. Please try to avoid light to slow down fluorescence quenching;
④ For your safety and health, please wear a lab coat and disposable gloves.
