Oregon green 488 azide (Difluorocarboxyfluorescein azide, 6-isomer) is a bright green fluorescent azide-activated probe that reacts with terminal alkynes via copper-catalyzed azide-alkyne cycloaddition (CuAAC). Oregon green 488 azide can label goat anti-mouse IgG and exhibits excellent luminescence efficiency. Oregon green 488 azide, as a streptavidin conjugate, is used for flow cytometry staining of macrophages [1] [2].
In Vitro, Guidelines (The following is our recommended protocol, which serves only as a guideline and should be modified according to your specific needs). 1. Materials List: Cells: Fixed cell samples treated with alkyne-containing probes (e.g., EdU, AHA, alkyne fatty acids, etc.). 2. Staining Reagents: Oregon Green 488 azide Click reaction buffer (typically PBS, pH ~7.4) Copper catalyst solution (e.g., CuSO4, usually at a concentration of 1-10 mM) Reductant/ligand solution (e.g., sodium ascorbate, usually at a concentration of 20-100 mM; or TBTA ) Wash solution: PBS Blocking solution: PBS containing 1-5% BSA Equipment: Light-proof humidified chamber, pipettes, centrifuge, fluorescence microscope or flow cytometer. 3. Experimental Procedures 1.1 Sample Preparation Fixation and permeabilization: Follow your standard experimental protocol to fix cells with an appropriate fixative (e.g., 4% paraformaldehyde ) and treat them with a permeabilizing agent (e.g., PBS solution containing 0.1-0.5% Triton X-100) to allow dyes and reaction reagents to enter the cells. Blocking: Block the samples with blocking solution at room temperature for 30-60 minutes to reduce non-specific binding. 1.2 Preparation of Click Reaction Mixture (Perform in the Dark) Prepare a stock solution of Oregon Green 488 azide (e.g., 1-10 mM) using anhydrous DMSO (or other recommended solvents) under light-proof conditions. Dilute it before use. Prepare the click reaction working solution. A typical formulation for a 100 µL reaction system is as follows: Click reaction buffer (e.g., PBS) to make up the total volume to 100 µL Oregon Green 488 azide at a final concentration of 1-10 µM CuSO4 at a final concentration of 0.1-1.0 mM Reductant/ligand at a final concentration of 1-10 mM (if sodium ascorbate is used, the concentration is usually 5-10 mM) Note: If using a pre-mixed kit containing TBTA, follow its specific formulation. TBTA can improve reaction efficiency and reduce copper toxicity. 1.3 Click Reaction Labeling Remove the blocking solution from the samples. Cover the samples with freshly prepared click reaction working solution. Place the samples in a light-proof humidified chamber and incubate at room temperature for 30 minutes to 2 hours. The specific incubation time needs optimization. 1.4 Washing Remove the reaction solution after incubation. Wash the samples with PBS 3-4 times, 5-10 minutes each time, to thoroughly remove unreacted dyes and copper catalyst. 1.5 Counterstaining and Mounting (For Imaging) An appropriate nuclear dye (e.g., DAPI, Hoechst) can be optionally used for counterstaining. Mount the samples with a suitable anti-fade mounting medium (e.g., ProLong, Vectashield). 1.6 Detection Analyze the samples using a fluorescence microscope or flow cytometer. Key Notes: Light protection: Strict light protection is required throughout the process to prevent fluorescence quenching. Optimization: For the first experiment, optimize the dye concentration, reaction time and copper ion concentration to balance labeling efficiency and background signal. Control setup: The following controls must be set up: Positive control: Samples treated with alkyne-containing probes. Negative control 1: Samples treated with alkyne-containing probes but without the dye in the click reaction solution. Negative control 2: Samples not treated with alkyne-containing probes but subjected to the complete click reaction. Copper toxicity: When labeling live cells, high concentrations of copper ions may be cytotoxic. Consider using more biocompatible copper-free click chemistry (e.g., the reaction between cyclooctyne DBCO and azide), but in this case, DBCO-conjugated Oregon Green 488 instead of the azide form is required.
References:
[1]. Panchuk-Voloshina N, et al. Alexa dyes, a series of new fluorescent dyes that yield exceptionally bright, photostable conjugates. J Histochem Cytochem. 1999;47(9):1179-1188.
[2]. Schwamborn M, et al. Monitoring ATPase induced pH changes in single proteoliposomes with the lipid-coupled fluorophore Oregon Green 488. Analyst. 2017;142(14):2670-2677.
In Vitro, Guidelines (The following is our recommended protocol, which serves only as a guideline and should be modified according to your specific needs). 1. Materials List: Cells: Fixed cell samples treated with alkyne-containing probes (e.g., EdU, AHA, alkyne fatty acids, etc.). 2. Staining Reagents: Oregon Green 488 azide Click reaction buffer (typically PBS, pH ~7.4) Copper catalyst solution (e.g., CuSO4, usually at a concentration of 1-10 mM) Reductant/ligand solution (e.g., sodium ascorbate, usually at a concentration of 20-100 mM; or TBTA ) Wash solution: PBS Blocking solution: PBS containing 1-5% BSA Equipment: Light-proof humidified chamber, pipettes, centrifuge, fluorescence microscope or flow cytometer. 3. Experimental Procedures 1.1 Sample Preparation Fixation and permeabilization: Follow your standard experimental protocol to fix cells with an appropriate fixative (e.g., 4% paraformaldehyde ) and treat them with a permeabilizing agent (e.g., PBS solution containing 0.1-0.5% Triton X-100) to allow dyes and reaction reagents to enter the cells. Blocking: Block the samples with blocking solution at room temperature for 30-60 minutes to reduce non-specific binding. 1.2 Preparation of Click Reaction Mixture (Perform in the Dark) Prepare a stock solution of Oregon Green 488 azide (e.g., 1-10 mM) using anhydrous DMSO (or other recommended solvents) under light-proof conditions. Dilute it before use. Prepare the click reaction working solution. A typical formulation for a 100 µL reaction system is as follows: Click reaction buffer (e.g., PBS) to make up the total volume to 100 µL Oregon Green 488 azide at a final concentration of 1-10 µM CuSO4 at a final concentration of 0.1-1.0 mM Reductant/ligand at a final concentration of 1-10 mM (if sodium ascorbate is used, the concentration is usually 5-10 mM) Note: If using a pre-mixed kit containing TBTA, follow its specific formulation. TBTA can improve reaction efficiency and reduce copper toxicity. 1.3 Click Reaction Labeling Remove the blocking solution from the samples. Cover the samples with freshly prepared click reaction working solution. Place the samples in a light-proof humidified chamber and incubate at room temperature for 30 minutes to 2 hours. The specific incubation time needs optimization. 1.4 Washing Remove the reaction solution after incubation. Wash the samples with PBS 3-4 times, 5-10 minutes each time, to thoroughly remove unreacted dyes and copper catalyst. 1.5 Counterstaining and Mounting (For Imaging) An appropriate nuclear dye (e.g., DAPI, Hoechst) can be optionally used for counterstaining. Mount the samples with a suitable anti-fade mounting medium (e.g., ProLong, Vectashield). 1.6 Detection Analyze the samples using a fluorescence microscope or flow cytometer. Key Notes: Light protection: Strict light protection is required throughout the process to prevent fluorescence quenching. Optimization: For the first experiment, optimize the dye concentration, reaction time and copper ion concentration to balance labeling efficiency and background signal. Control setup: The following controls must be set up: Positive control: Samples treated with alkyne-containing probes. Negative control 1: Samples treated with alkyne-containing probes but without the dye in the click reaction solution. Negative control 2: Samples not treated with alkyne-containing probes but subjected to the complete click reaction. Copper toxicity: When labeling live cells, high concentrations of copper ions may be cytotoxic. Consider using more biocompatible copper-free click chemistry (e.g., the reaction between cyclooctyne DBCO and azide), but in this case, DBCO-conjugated Oregon Green 488 instead of the azide form is required.
References:
[1]. Panchuk-Voloshina N, et al. Alexa dyes, a series of new fluorescent dyes that yield exceptionally bright, photostable conjugates. J Histochem Cytochem. 1999;47(9):1179-1188.
[2]. Schwamborn M, et al. Monitoring ATPase induced pH changes in single proteoliposomes with the lipid-coupled fluorophore Oregon Green 488. Analyst. 2017;142(14):2670-2677.