Rhod-4, AM, Cell Permeant (Synonyms: Rhod-4,Acetoxymethyl Ester)

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

1. Preparation of staining solution

(1) Prepare storage solution: Dissolve Rhod-4 AM in DMSO and prepare a storage solution with a concentration of 1-10mM.

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 1-10μM.

(Optional) If the efficiency of Rhod-4 AM entering cells is unsatisfactory, an appropriate amount of 20% Pluronic F-127 solution can be added to the Rhod-4 AM solution. This prevents Rhod-4 AM aggregation in the buffer and enhances its cell entry, with the final concentration of Pluronic F-127 controlled between 0.02-0.05%.

Note: To prepare a 20% (w/v) Pluronic F-127 DMSO stock solution: Dissolve 100mg of Pluronic F-127 (Cat.No.：GB30090) in 0.5 mL DMSO to create a 20% (w/v) DMSO stock solution. The dissolution requires heating at 40-50°C for 20-30 minutes. Once dissolved, store it at room temperature and avoid refrigeration. If crystallization occurs, it can be re-dissolved by heating without affecting its use.

Pluronic F-127 can reduce the stability of Rhod-4 AM, so it is only recommended to be added during the preparation of the working solution and not advised to be included in the storage solution.

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

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 Rhod-4 AM working solution to resuspend the cells, and incubate at 37 °C in the dark for 0.5-1h. 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 cells in pre-warmed serum-free cell culture medium or PBS and observe by fluorescence microscope 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 100 μL of dye working solution from one corner of the coverslip and shake gently to evenly cover all cells with the dye.

(4) Incubate at 37 °C in the dark for 0.5-1h. 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 wavelength of Rhod-4 AM is 530/555nm.

(Optimization) If the initial experiment does not yield a clear determination of the optimal incubation temperature and duration, it is recommended to try incubating at 37°C for 30 minutes and then observe the fluorescence results. If there is significant cell death, consider shortening the incubation time or lowering the temperature. Conversely, if the fluorescence intensity is too weak, appropriately extend the incubation time.

Precautions:

1) Fluorescent dyes all have quenching problems. Please avoid light as much as possible to slow down fluorescence quenching.

2) For your safety and health, please wear a lab coat and disposable gloves.