HKSOX-1m (5/6-mixture) hydrobromide is a O 2 fluorescent probe for mitochondria-targeting (Ex/Em=509/534nm; green), exhibiting excellent selectivity and sensitivity toward O 2 over a broad range of pH, strong oxidants, and abundant reductants found in cells [1].
In Vitro, HKSOX-1m (5/6-mixture) hydrobromide (10 μM) sensitively captures signals for basal and Antimycin A (1 μM)-stimulated mitochondrial O2•− in differentiated human THP-1 cells, at very low laser power output[1]. Guidelines (Following is our recommended protocol. This protocol only provides a guideline, and should be modified according to your specific needs). 1. Preparation of HKSOX-1m (5/6-mixture) hydrobromide working solution 1.1 Preparation of the stock solution Dissolve HKSOX-1m (5/6-mixture) hydrobromide in DMSO to obtain 10 mM of stock solution. Note: It is recommended to store the stock solution at -20°C -80°C away from light and avoid repetitive freeze-thaw cycles. 1.2 Preparation of HKSOX-1m (5/6-mixture) hydrobromide working solution Dilute the stock solution in serum-free cell culture medium or PBS to obtain 1-10 μM of working solution. Note: Please adjust the concentration of HKSOX-1m (5/6-mixture) hydrobromide working solution according to the actual situation. 2. Cell staining (Suspension cells) 2.1 Centrifuge at 1000 g at 4°C for 3-5 minutes and then discard the supernatant. Wash twice with PBS, 5 minutes each time. The cell density is 1×106/mL 2.2 Add 1 mL of working solution, and then incubate at room temperature for 5-30 minutes. 2.3 Centrifuge at 400 g at 4°C for 3-4 minutes and then discard the supernatant. 2.4 Wash twice with PBS, 5 minutes each time. 2.5 Resuspend cells with serum-free cell culture medium or PBS. Observation by fluorescence microscopy or flow cytometry. 3. Cell staining (Adherent cells) 3.1 Culture adherent cells on sterile coverslips. 3.2 Remove the coverslip from the medium and aspirate excess medium. 3.3 Add 100 μL of working solution, gently shake it to completely cover the cells, and then incubate at room temperature for 5-30 minutes. 3.4 Wash twice with medium, 5 minutes each time. Observation by fluorescence microscopy or flow cytometry.
References:
[1]. Jun Jacob Hu, et al. Fluorescent Probe HKSOX-1 for Imaging and Detection of Endogenous Superoxide in Live Cells and In Vivo. J Am Chem Soc. 2015 Jun 3;137(21):6837-43.
In Vitro, HKSOX-1m (5/6-mixture) hydrobromide (10 μM) sensitively captures signals for basal and Antimycin A (1 μM)-stimulated mitochondrial O2•− in differentiated human THP-1 cells, at very low laser power output[1]. Guidelines (Following is our recommended protocol. This protocol only provides a guideline, and should be modified according to your specific needs). 1. Preparation of HKSOX-1m (5/6-mixture) hydrobromide working solution 1.1 Preparation of the stock solution Dissolve HKSOX-1m (5/6-mixture) hydrobromide in DMSO to obtain 10 mM of stock solution. Note: It is recommended to store the stock solution at -20°C -80°C away from light and avoid repetitive freeze-thaw cycles. 1.2 Preparation of HKSOX-1m (5/6-mixture) hydrobromide working solution Dilute the stock solution in serum-free cell culture medium or PBS to obtain 1-10 μM of working solution. Note: Please adjust the concentration of HKSOX-1m (5/6-mixture) hydrobromide working solution according to the actual situation. 2. Cell staining (Suspension cells) 2.1 Centrifuge at 1000 g at 4°C for 3-5 minutes and then discard the supernatant. Wash twice with PBS, 5 minutes each time. The cell density is 1×106/mL 2.2 Add 1 mL of working solution, and then incubate at room temperature for 5-30 minutes. 2.3 Centrifuge at 400 g at 4°C for 3-4 minutes and then discard the supernatant. 2.4 Wash twice with PBS, 5 minutes each time. 2.5 Resuspend cells with serum-free cell culture medium or PBS. Observation by fluorescence microscopy or flow cytometry. 3. Cell staining (Adherent cells) 3.1 Culture adherent cells on sterile coverslips. 3.2 Remove the coverslip from the medium and aspirate excess medium. 3.3 Add 100 μL of working solution, gently shake it to completely cover the cells, and then incubate at room temperature for 5-30 minutes. 3.4 Wash twice with medium, 5 minutes each time. Observation by fluorescence microscopy or flow cytometry.
References:
[1]. Jun Jacob Hu, et al. Fluorescent Probe HKSOX-1 for Imaging and Detection of Endogenous Superoxide in Live Cells and In Vivo. J Am Chem Soc. 2015 Jun 3;137(21):6837-43.