4,5-Diaminofluorescein

4,5-Diaminofluorescein is a fluorescent detector for nitric oxide (NO) in cells and tissues^[1]. In Vitro: 4,5-Diaminofluorescein Experimental Procedure^[2]
1. Preparation
1.1 Solution Preparation:
1) L-Arginine Stock Solution: Dissolve L-Arginine in water to make a 100 mM solution (0.021 g/ml).
2) A23187 Stock Solution: Dissolve 1 mg A23187 in 191 µL DMSO to make a 10 mM solution and store at -20°C. 3) DAF-2 Stock Solution: The product is shipped as a 5 mM DMSO solution and stored frozen at -80°C after aliquoting.
4) PBS+Ca²⁺ solution: KH₂PO₄ 1.47 mM; Na₂HPO•7H₂O 9.57 mM; NaCl 137.00 mM; MgSO₄•7H₂O 0.49mM; CaCl₂•2 H₂O 0.90 mM. Dissolve CaCl₂ in 1L of water and other ingredients in another 1L of water. Carefully mix the two solutions and adjust the pH to 7.4. Since the fluorescence intensity of DAF derivatives is related to the pH value, the pH value needs to be strictly controlled.
1.2 Pre-experimental preparation:
1) Preheat a water bath and a desiccator/incubator to 37°C.
2) Add L-arginine stock solution (100mM) to a final concentration of 100 µM in PBS+Ca²⁺ solution at a ratio of 1:1000 and preheat the solution in a 37°C water bath.
3) Dilute A23187 stock solution (10 mM) 1:5 with DMSO. Dilute DAF-2 stock solution (5 mM) 1:250 with L-arginine in PBS+Ca²⁺ solution and store on ice in a light-proof box until use.
4) Start the spectrofluorometer software. In a dark room, bring the cuvette, rinse water, and waste container to the spectrofluorometer to prepare the sample for processing.
Note: All operations involving DAF-2 must be performed in a dark room! During the transportation of samples to the spectrofluorometer, be sure to protect the samples from light!
2. Formal Experiment:
1) EA.hy 926 Cell Culture
2) Remove the supernatant of EA.hy 926 cells and wash the cells twice with 1 mL PBS+Ca²⁺ solution.
3) Incubate the cells with 2 mL PBS+Ca²⁺ solution supplemented with L-arginine for 5 min at 37°C to allow the cells to reach equilibrium with the specified concentration of L-arginine.
4) Add 1 µL of diluted A23187 solution (final concentration is 1 µM). Add 10 µL of the diluted DAF-2 solution (final concentration 0.1 µM). Incubate the cells again at 37°C in the dark for 5 min, during which time the released NO will react with DAF-2 to form DAF-2T.
5) Quickly transfer the cell supernatant to a 2 ml plastic cap, ensuring that the reaction time is consistent for all samples.
6) Measure the fluorescence intensity of the cell supernatant in a standard cuvette as quickly as possible. Ensure that all samples are well protected from light until the measurement is completed. When measuring, the excitation wavelength should be between 490-495nm (need to be verified on a case-by-case basis), the emission wavelength is 515 nm, the emission and excitation slit widths are both 10nm, and the sensitivity should be selected at the highest setting possible. Also measure the wells without cells to calculate the autofluorescence of DAF-2.