NucPE1 (Nuclear Peroxy Emerald 1) (Synonyms: Nuclear Peroxy Emerald 1)

This plan only provides a guide, please modify it to meet your specific needs.
1. Prepare NucPE1 (Nuclear Peroxy Emerald 1) staining solution
(1) Prepare dye stock solution: Use DMSO to dissolve NucPE1 into a 5-10mM stock solution.
Note: Unused stock solution should be aliquoted and stored in the dark at -20°C to avoid repeated freezing and thawing.
(2) Prepare dye working solution: Dilute the stock solution with a suitable buffer (such as serum-free medium, HBSS or PBS) to prepare a NucPE1 working solution with a concentration of 1-10 μM.
Note: It is recommended that unused stock solutions be aliquoted and stored in the dark at -4°C or -20°C, and avoid repeated freezing and thawing.
2. Staining of adherent cells
(1) Cell plating is performed the day before using fluorescent dyes.
(2) After treating the cells with experimental drugs/treatments, wash the cells 2-3 times with PBS.
(3) Add preheated NucPE1 dye working solution and incubate at 37°C in the dark for 15-30 minutes. The optimal incubation time for different cells is different, please explore by yourself according to the specific experimental needs.
(4) After incubation, wash with PBS 2-3 times for 5 minutes each time.
(5) Cover the cells with pre-warmed serum-free cell culture medium or PBS (keep the cells moist).
3. Cell suspension staining
(1) Suspension cells: Centrifuge the suspended cells at 4°C, 1000g for 3-5 minutes, discard the supernatant, and wash twice with PBS for 5 minutes each time.
(2) Adherent cells: After washing with PBS 2-3 times, use trypsin to digest the cells, centrifuge at 1000g for 3-5 minutes at 4°C, and discard the supernatant.
(3) Resuspend approximately 1x106 cells in 0.5-1mL working solution and incubate at 37°C in the dark for 15-30 minutes. The optimal incubation time for different cells is different, please explore by yourself according to the specific experimental needs.
4. Fluorescence detection: Use confocal microscopy or flow cytometry for analysis[1]. Live imaging of the NucPE1 probe was performed using an argon laser excited at 488nm and emissions collected using a META detector at approximately 520nm; when multiple stainings were performed with NucPE1 and PO1, a multi-track scan mode was applied to collect images[2].

Note:
1) Fluorescent dyes have quenching problems, please try to avoid light to slow down fluorescence quenching.
2) For your safety and health, please wear a lab coat and disposable gloves for operation.
References:
[1]. Stanicka J, et al. NADPH oxidase-generated hydrogen peroxide induces DNA damage in mutant FLT3-expressing leukemia cells. J Biol Chem. 2015 Apr 10;290(15):9348-61.
[2]. Dickinson BC, et al. A nuclear-localized fluorescent hydrogen peroxide probe for monitoring sirtuin-mediated oxidative stress responses in vivo. Chem Biol. 2011 Aug 26;18(8):943-8.