Fluo-4 AM (Synonyms: Fluo-4 Acetoxymethyl ester)

This program only provides a guide, please modify according to your specific needs.
1. Prepare dyeing solution
(1) Configuration storage solution: AM ester storage solution with a concentration of 1-10mm was prepared in anhydrous DMSO.
Notes:
② Unused storage solution is stored away from light at -20°C or -80°C after packaging to avoid repeated freezing and thawing;
② Fluo-4AM is easy to absorb moisture, please put it in a dry environment to room temperature before opening it. Centrifuge it briefly before opening to ensure that the powder falls to the bottom of the tube.
(2) Configuration of the working liquid: Dilute the storage solution with a suitable buffer (such as: no serum and phenol red medium or PBS), and prepare a working liquid with a concentration of 1-10μM.
Notes:
(1) When preparing the Fluo-4 AM dyeing solution, an appropriate amount of 20% Pluronic F-127 solution should be added to the storage solution to enhance the water solubility of the AM probe;
②Pluronic F-127 can prevent Fluo-4 AM from accumulating in solution and promote better probe entry into cells. However, PluronicF-127 can reduce the stability of AM probes, so it is only recommended to add PluronICF-127 in the preparation of working solution, and not to add storage solution for long-term preservation.
③ Prepare 20%(w/v) Pluronic F-127 mother liquor: weigh 100mg Pluronic F-127 powder (article number: GB30090), add 500μL DMSO, heat at 40-50℃ for 20-30min, and store at room temperature. If there is crystallization can be reheated to dissolve, does not affect the use;
④ (Optional) GLPBIO provides dissolved Pluronic F-127(20% Solution in DMSO), product number: GB30091;
Add an equal volume of 20% Pluronic F-127 solution to the storage solution so that the final working concentration of Pluronic F-127 is about 0.02%;
Please adjust the concentration of the working liquid according to the actual situation, and use it now to avoid repeated freezing and thawing.
2. Cell suspension staining
(1) Suspension cells: Centrifuge at 4°C, 1000g, for 3-5 minutes, discard the supernatant, and clean with PBS or other buffers twice, each time for 5 minutes;
(2) Adherent cells: Wash with PBS or other buffers twice, add pancreatic enzyme digestion cells, and centrifuge 1000g for 3-5min after digestion;
(3) The cells were re-suspended with dye working solution and incubated at or below room temperature in the dark for 20min-2h. The best incubation time of different cells is different, please explore by yourself according to the specific experimental requirements;
Notes:
① The recommended working concentration of AM ester dyes in most cells is 4-5μM, and the specific concentration should be optimized according to the experimental requirements. In order to avoid cytotoxicity caused by overloading, it is recommended to use the lowest probe concentration as far as possible on the basis of obtaining effective results.
② (Optional) If the cell contains an organic anion transporter, it may be necessary to add prosulfasol (GC16825, Probenecid, 1-2.5mM) or sulpyrone (GC11049, Sulfinpyrazone, 0.1-0.25mM) to the cell medium to reduce the leakage level of the de-esterification probe. The storage solution is alkaline, so the pH needs to be re-adjusted after adding the medium;
③ If the medium containing serum is used, the serum lactase will degrade AM, thus reducing the dye loading effect; The culture medium containing phenol red will make the background value slightly higher, it is recommended to wash the cells with the non-indicator culture medium for 2 to 3 times before adding the dyeing solution.
④ Reducing the loading temperature of the probe may reduce the localization of the probe.
(4) After incubation, centrifuge 1000g for 5 minutes, remove the staining solution, add PBS or other buffer to clean 2-3 times, and remove the residual probe;
(5) Incubate at room temperature for 30min to ensure complete de-esterification of AM in cells.
3. Cell adhesion staining
(1) The adherent cells were cultured on a sterile cover slide;
(2) Remove the cover glass from the medium, suck out the excess medium, and place the cover glass in a humid environment;
(3) Add 100μL of dye working liquid from one corner of the cover glass, and gently shake the dye to evenly cover all cells;
(4) Incubation at or below room temperature in the dark for 20min-2h. The best incubation time of different cells is different, please explore by yourself according to the specific experimental requirements;
(5) After the end of incubation, absorb and discard the dye working solution, and clean the cover glass with PBS or other buffer solution for 2 to 3 times;
(6) Incubate at room temperature for 30min.
4. Microscope detection: the maximum excitation/emission wavelength of Fluo-4AM is 494/506 nm.
Notes:
(1) It is recommended to add 20% Pluronic F-127 solution when configuring the working fluid, and the addition of Pluronic F-127 can increase the dispersibility of Fluo-4 AM in the aqueous solution.
(2) The release of Fluo-4 AM could be reduced by adding prosulfazol (GC16825, Probenecid, 1-2.5mM) or sulpiridone (GC11049, Sulfinpyrazone, 0.1-0.25mM) into the cell culture medium. The storage solution is alkaline, so the pH needs to be re-adjusted after the medium is added.
(3) F luo-4 AM is easy to absorb moisture. After taking it out of the refrigerator, please make sure to put it in a dry environment to room temperature before opening it. Due to the small amount of reagent, please centrifuge it briefly before opening to ensure that the powder falls into the bottom of the tube.
(4) F luo-4 AM mother liquor is very easy to decompose in water, if it cannot be used at one time, it is recommended to pack and store, for example, pack into 5μl/tube, seal with a sealing film, wrap with aluminum foil, put in a plastic bag with good airtight performance, and put in a bag of desiccant, sealed at ≤-20 ° C and store away from light.
(5) Before the formal experiment, it is recommended that you explore the number of cells, the final concentration of calcium ion fluorescence probe, culture time, etc., to find the appropriate experimental conditions.
(6) Fluorescent dyes have quenching problems, please pay attention to avoid light as much as possible to slow down fluorescence quenching.
(7) Before fluorescence incubation, cells should be washed in an indicator free medium to remove non-specific dye binding to the cell surface.