Evaluation of calcium content using Mag-Fura-2 AM[1]
This protocol only provides a guideline, and should be modified according to your specific needs
1. Prepare dyeing solution
(1) Prepare stock solution: Prepare Mag-Fura-2 AM stock solution with a concentration of 1-10mM in high-quality anhydrous DMSO. Notice: Aliquot the unused stock solution and store it in the dark at -20°C or -80°C to avoid repeated freezing and thawing.
(2) Prepare working solution: Dilute the stock solution with a suitable buffer (such as serum- and phenol red-free culture medium or PBS) to prepare a working solution with a concentration of 1-10μM. Please adjust the concentration of the working fluid according to the actual situation, and prepare it now to avoid repeated freezing and thawing. Notice: The working solution must be prepared fresh on the day of the experiment. Before use, Mag-Fura-2 AM solution needs to be rewarmed and centrifuged.
2. Cell suspension staining
(1) Suspension cells: centrifuge at 4°C and 1000g for 3-5 minutes, discard the supernatant, and wash twice with PBS or other buffer, 5 minutes each time;
(2) Adherent cells: wash twice with PBS or other buffers, add trypsin to digest the cells, and centrifuge at 1000g for 3-5 minutes after digestion is completed;
(3) Incubate the cells at the required temperature (25°C-37°C) for 10 minutes to allow the ionic gradient to reach stability and equilibrium. Take 4μl of the Mag-Fura-2 AM stock solution and 5μl of 20% Pluronic® F-127, add them to 1ml of cell culture medium, and vortex-mix vigorously to obtain a homogeneous probe dispersion solution. Take 250μl of the prepared probe dispersion solution and add it to 750μl of cell-containing culture medium, mix well. At this point, a working solution with a final concentration of 1-5μM is obtained based on the initial mother solution concentration. Incubate the probe for 15-60 minutes according to the requirements. Wash the cells 3 times, and then continue to incubate for 30 minutes to ensure that the probes in the cells are completely de-esterified.
Note:
1) The recommended working concentration of Mag-Fura-2 AM in most cells is 1-5μM, and the specific concentration needs to be optimized according to experimental requirements. In order to avoid cytotoxicity caused by overloading, use the lowest probe concentration possible based on achieving effective results;
2) (Optional) If the cells contain organic anion transporters, you may need to add probenecid (1-2.5mM) or sulfinpyrazone (0.1-0.25mM) to the cell culture medium. to reduce the leakage level of the de-esterification probe. The stock solution of probenecid or sulfinpyrazone is alkaline, so the pH needs to be readjusted after adding the culture medium;
3) If serum-containing medium is used, serum lactonase will degrade Mag-Fura-2 AM, thereby reducing the dye loading effect; while phenol red-containing medium will make the background value slightly higher. Wash the cells 2 to 3 times before adding the staining working solution;
4) Lowering the probe loading temperature may reduce the compartmentalization of the probe.
(4) Select the appropriate instrument and conduct fluorescence measurement (Ex/Em=336/503nm).
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) Incubate the cells at the required temperature (25°C-37°C) for 10 minutes to allow the ionic gradient to reach stability and equilibrium. Take 4μl of the Mag-Fura-2 AM stock solution and 5μl of 20% Pluronic® F-127, add them to 1ml of cell culture medium, and vortex-mix vigorously to obtain a homogeneous probe dispersion solution. Take 250μl of the prepared probe dispersion solution and add it to 750μl of cell-containing culture medium, mix well. At this point, a working solution with a final concentration of 1-5μM is obtained based on the initial mother solution concentration. Incubate the probe for 15-60 minutes according to the requirements. Wash the cells 3 times, and then continue to incubate for 30 minutes to ensure that the probes in the cells are completely de-esterified.
(4) Select the appropriate instrument and conduct fluorescence measurement (Ex/Em=336/503nm).
4. 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 when operating
(3) Mag-Fura-2 AM should be stored in the dark throughout the experiment.
(4) This protocol and all reagents are for scientific research use only.
References:
[1] Gurney A M, Drummond R M, Fay F S. Calcium signalling in sarcoplasmic reticulum, cytoplasm and mitochondria during activation of rabbit aorta myocytes[J]. Cell Calcium, 2000, 27(6): 339-351. |
