Filipin III

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

1. Preparation of cell membrane staining solution

(1) Prepare DMSO storage solution: Use DMSO or absolute ethanol to dissolve Filipin III and prepare a storage solution with a concentration of 1-5mM.

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 an appropriate buffer (such as serum-free medium or PBS) and prepare a working solution with a concentration of 1-250 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 1 mL of Filipin III working solution to resuspend the cells and incubate at room temperature in the dark for 15-30 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 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 room temperature in the dark for 15-30 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 excitation light range of Filipin III is 340-380nm, and the emission light range is 385-470 nm.

Precautions:

1) Note：Filipin fluorescent staining photobleaches very rapidly, thus the sample should be analyzed immediately.

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