MQAE (Synonyms: MQAE)

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

1. Prepare MQAE staining solution:

(1) Prepare dye storage solution: Dissolve MQAE in a mixture of acetonitrile and water (1:1), prepare a 500 mM stock solution, and use a 0.22 μm filter to filter and sterilize. The stock solution was aliquoted and stored in the dark at -20 °C.

(2) Use Krebs-hepes buffer to dilute the stock solution to obtain an MQAE working solution with a concentration of 5-10 mM.

Krebs-hepes buffer: 20mM HEPES, 128mM NaCl, 2.5mM KCl, 2.7mM CaCl₂, 1mMMgCl₂, 16mM glucose, pH 7.4

Note: Please adjust the concentration of MQAE working fluid 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 Krebs-hepes buffer for 5 minutes each time.

(2) Wash the adherent cells twice with Krebs-hepes buffer, 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 30-60 minutes. 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) Resuspend the cells in serum-free cell culture medium or PBS and observe through a microscope.

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 30-60 minutes.

(4) Aspirate away the dye working solution and wash the coverslip with PBS 2 to 3 times for 5 minutes each time.

4. Microscope detection: Use an optical microscope or flow cytometer to detect stained cells. The maximum excitation/emission light of MQAE is 350/460 nm.

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]. Chenchen Ma,et. Colloidal Particles in Tuna Head Soup: Chemical Localization, Structural Change, and Antioxidant Property. 2021 Mar 29:8:638390. doi: 10.3389/fnut.2021.638390. eCollection 2021.

[2]. Yukiko Ikeuchi,et. Measurement of [Cl-]i unaffected by the cell volume change using MQAE-based two-photon microscopy in airway ciliary cells of mice. 2018 Mar;68(2):191-199. doi: 10.1007/s12576-018-0591-y. Epub 2018 Jan 13.