This plan only provides a guide, please modify it to meet your specific needs.
1. Prepare dyeing solution
(1) Dye stock solution: Use DMSO to dissolve 2-Di-1-ASP into a 1-5mM stock solution. The prepared stock solution is aliquoted and stored in the dark at -20 or -80°C.
(2) Dye working solution: Dilute the stock solution with a suitable buffer (such as serum-free medium, HBSS or PBS) to prepare an 2-Di-1-ASP working solution with a concentration of 1-5μM.
Note: Please adjust and optimize the working fluid concentration according to the actual situation, and prepare it now.
2. Cell suspension staining (taking 6-well plate as an example)
(1) Centrifuge suspended cells at 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.
(3) Add 1 mL of dye working solution to resuspend the cells, and incubate at room temperature in the dark for 15-30minutes. The optimal culture time for different cells is different.
(4) After the incubation, centrifuge at 1000g for 5 minutes, remove the supernatant, and add PBS to wash 2-3 times, 5 minutes each time.
(5) Use serum-free cell culture medium or PBS to resuspend the cells and observe them through 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 100 μL of dye working solution from one corner of the coverslip, shake gently to evenly cover all cells with the dye, and incubate at room temperature in the dark for 15-30 minutes.
(4) Aspirate away the dye working solution and use culture solution to wash the coverslip 2 to 3 times for 5 minutes each time.
4. Microscope detection: The maximum excitation light/emission light of 2-Di-1-ASP is 485/607nm.
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.
References:
[1].Yu-Meng Wang, et, al. MeCP2 duplication causes hyperandrogenism by upregulating LHCGR and downregulating RORα. 2021 Oct 25;12(11):999. doi: 10.1038/s41419-021-04277-4.
