4,5-Diaminofluorescein

Guide (The following is the experimental plan we recommend. This plan serves only as a reference guide. The specific operations should be adjusted according to your actual needs.)
1. Preparatory Work 1.1 Solution Preparation: 1) L-Arginine stock solution: Dissolve L-Arginine in water to prepare a 100 mM solution (0.021 g/mL).
2) A23187 stock solution: Dissolve 1 mg of A23187 in 191 μL of DMSO to prepare a 10 mM solution, which should be stored at -20°C.
3) DAF-2 stock solution: During transportation, it is a 5 mM DMSO solution. After being aliquoted, it is stored frozen at -80°C.
4) PBS + Ca²⁺ solution: KH₂PO₄ 1.47 mM; Na₂HPO?7H₂O 9.57 mM; NaCl 137.00 mM; MgSO₄?7H₂O 0.49 mM; CaCl₂?2 H₂O 0.90 mM.
Dissolve CaCl₂ in 1 liter of water, and dissolve the other components in another 1 liter of water. Carefully mix the two solutions and adjust the pH to 7.4, as the fluorescence intensity of the DAF derivative is related to the pH value, so the pH value must be strictly controlled.
1.2 Preparations before the experiment: Preheat the water bath and the drying box / incubator to 37°C.
2) Add the L-arginine stock solution (100 mM) to the PBS + Ca²⁺ solution, adjusting the final concentration to 100 μM. This is done by adding at a ratio of 1:1000. Then, preheat the solution in a 37°C water bath.
3) Dilute the A23187 stock solution (10 mM) with DMSO at a ratio of 1:5. Dilute the DAF-2 stock solution (5 mM) with PBS + Ca²⁺ solution containing L-arginine at a ratio of 1:250, and store it in a light-proof box on ice until it is ready for use.
4) Start the fluorescence spectrophotometer software. In the darkroom, bring the cuvette, rinsing water and waste container to the fluorescence spectrophotometer, and prepare to process the samples.
Note: All operations involving DAF-2 must be conducted in a darkroom! During the transportation of the samples to the fluorescence spectrophotometer, make sure to keep the samples away from light!
2. Official Experiment: 1) Cultivation of EA.hy 926 cells 2) Remove the supernatant from the EA.hy 926 cells and wash the cells twice with 1 mL of PBS + Ca²⁺ solution.
3) The cells were incubated with 2 mL of PBS + Ca²⁺ solution containing L-arginine for 5 minutes at a temperature of 37°C, allowing the cells to reach an equilibrium state with the specific concentration of L-arginine.
4) Add 1 μL of the diluted A23187 solution (final concentration of 1 μM). Add 10 μL of the diluted DAF-2 solution (final concentration of 0.1 μM). Once again, incubate the cells in a 37°C dark environment for 5 minutes. During this period, the released NO will react with DAF-2 to form DAF-2T.
5) Quickly transfer the cell supernatant to a 2 ml plastic tube, ensuring that the reaction time for all samples is consistent.
6) Measure the fluorescence intensity of the cell supernatant in a standard cuvette as soon as possible. Before the measurement is completed, ensure that all samples are well protected from light. During the measurement, the excitation wavelength should be between 490-495nm (this needs to be verified according to specific circumstances), the emission wavelength is 515nm, the width of the emission and excitation slits is 10nm, and the sensitivity should be set to the highest level possible. Also, measure the wells without cells to calculate the spontaneous fluorescence of DAF-2.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.