SynaptoRedTM C2 (Synonyms: FM4-64)

This protocol provides a guide only and should be modified according to your specific needs.

1. Preparation of cell membrane staining solution

(1) Prepare DMSO or EtOH storage solution: the storage solution is prepared with DMSO or EtOH, the concentration is 1~5mM.

Note: Store the unused stock solution in aliquots at -20°C or -80°C in the dark and avoid repeated freezing and thawing.

(2) Preparation of working solution: Dilute the stock solution with an appropriate buffer (such as: serum-free medium, HBSS or PBS) to prepare a working solution with a concentration of 0.5-5 μM.

Note: The final concentration of the working solution needs to be optimized according to different cell lines and experimental systems. It is recommended to start from the recommended concentration and explore the optimal concentration in the range of 10 times.

2. Cell Staining

(1). Suspension cells: Collect cells by centrifugation at 4°C, 1000-1500rpm, add PBS to wash twice, 5 minutes each time. The cells were resuspended in the TMA-DPH working solution with a final concentration of 2 μM, incubated at room temperature in the dark for a short period of time (10S), and then the unwashed cells were immediately plated and transferred to the microscope for observation^[1].

Adherent cells; discard medium and add trypsin to digest cells. After centrifuging at 4°C, 1000-1500rpm to discard the supernatant, add PBS to wash twice, 5 minutes each time.

(2). Adherent cells: Discard the medium and wash twice with PBS, 5 minutes each time.

1). Cell plasma membrane staining: Cells were incubated with TMA-DPH at a final concentration of 2 μM for a short period of time (10S) at room temperature. The unwashed slides were then transferred to a microscope for observation.

2). Cell internalized membrane marker staining: the cells were moved to a glass slide flask, and incubated at 37° with TMA-DPH at a final concentration of 2 μM for the required time according to the experimental requirements. After incubation, slides are washed in PBS with gentle shaking for a few seconds to remove peripheral labeling^[1].

4. Microscopic examination: the excitation/emission light of TMA-DPH is 355/430nm respectively.

Note:

1) Fluorescent dyes have quenching problems, please try to avoid light to slow down fluorescence quenching.

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

References:

[1]. Illinger, D., and Kurhy, J.G. The kinetic aspects of intracellular fluorescence labeling with TMA-DPH support the maturation model for endocytosis in L929 cells. Journal of Cell Biology 125(4), 783-794 (1994).