ER–Tracker Blue–White DPX

This plan only provides a guide, please modify it to meet your specific needs.

1. Preparation of staining solution

(1) This product is provided as a DMSO stock solution with a concentration of 1mM. Take out the frozen dyeing solution, return it to room temperature, and concentrate the dyeing solution at the bottom of the tube by brief centrifugation.

(2) It is recommended to use serum-free medium or PBS diluted stock solution preheated at 37°C to prepare a dye working solution with a concentration of 100nM-1μM.

Note: Please adjust the concentration of the working fluid according to the actual situation and prepare it now.

2. Cell suspension staining

(1) Centrifuge the suspended cells at 4°C and 1000g for 3-5 minutes, discard the supernatant, and wash twice with PBS for 5 minutes each time.

(2) Wash the adherent cells twice with PBS, add trypsin to digest the cells, and centrifuge at 1000g for 3-5 minutes after digestion is completed.

(2) Add 1 mL of dye working solution to resuspend the cells and incubate at room temperature in the dark for 5-30 minutes.

Note: The optimal culture time for different cells is different and can be adjusted according to specific experimental needs.

(3) After the incubation, centrifuge at 1000g for 5 minutes, remove the supernatant, add PBS and wash 2-3 times, 5 minutes each time.

(4) Resuspend 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, aspirate the excess culture medium, and place the coverslip in a humid environment.

(3) Add 100uL 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 culture time for different cells is different.

(5) After the incubation, discard the dye working solution and wash the coverslip 2 to 3 times with pre-warmed culture solution.

4. Microscope detection: The maximum excitation/emission light of ER–Tracker Blue–White DPX is 374/430-640nm respectively.

Precautions:

①    After formaldehyde fixation of stained cells for 10-20 minutes at 37℃, the ER–Tracker Blue–White DPX signal is only partially retained. If you want to fix stained cells, it is recommended to fix them with 4% formaldehyde at 37℃ for 2 minutes.

② If additional staining is required, it is recommended to permeabilize the cells with 0.2% Triton X-100 for 10 minutes.

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.