Hypochlorous acid (HOCl) is a critical member of the reactive oxygen species (ROS) produced by immune cells to fight infections. On the other hand, HOCl in homeostasis causes oxidative damage to biomolecules and is linked to many diseases, including inflammatory, neurodegenerative, and cardiovascular diseases. Herein, we detail a procedure for the preparation of a boron-dipyrromethene (BODIPY)-derived fluorescent probe for HOCl (BClO) and its application as an imaging reagent in living cells. BClO is synthesized in one pot through a four-step procedure that is nearly the same as that for conventional BODIPY dye preparation, except for the ratio of starting materials. BClO has an extremely rapid response (saturated within seconds) and is ultrasensitive to HOCl. The detection limit of BClO reaches the subnanomolar range, which is the highest HOCl sensitivity to date. Taking advantage of the ultrasensitive character of BClO, we have previously demonstrated its ability to detect endogenous HOCl generated by macrophages and shown that it can also be used to discriminate cancer cell lines (which show high HOCl production) from non-cancer cell lines (which show low HOCl production). The protocol requires ~2 d for probe synthesis and up to ~18 h for fluorescence imaging and flow cytometry assays.
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This work was financially supported by the National Science Foundation of China (21576037, 21406028, 21703025, and 21421005) and the NSFC-Liaoning United Fund (U1608222).
The authors declare no competing interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Key reference using this protocol
1. Zhu, H. et al. J. Am. Chem. Soc. 136, 12820–12823 (2014): https://doi.org/10.1021/ja505988g
Integrated supplementary information
NaOCl concentrations vary ranging from 0 to 6 µM. The treatment of HOCl elicits tiny changes in the absorbance of BClO.
NaOCl concentrations (from bottom to top): 0, 2, 4, 6, 8, 10, 12, 14, and 16 nM. Excitation at 480 nm.
(from left to right): 4.00, 4.53, 5.02, 5.52, 5.98, 6.51, 6.98, 7.40, 7.98, 8.51, and 9.02. Data represent mean values of fluorescence intensity ± standard derivation (n =3). Blue, red, and black dots represent data from 3 independent replicate experiments.
COS-7 (a) and HeLa (b) cells were incubated with BClO at 37 ºC, and fluorescence images were recorded at different time points: 0, 5, 10, and 20 min. After 20 min, BClO was completely activated by adding 12 µM of NaOCl. *: NaOCl (+). Scale bar is 200 µm. The intrinsic fluorescence of BClO in HeLa cells was higher than that in COS-7 cells, which is consistent with the result shown in Figure 4. After complete activation by NaOCl, BClO showed identical fluorescence in COS-7 and HeLa cells, suggesting its unbiased cell uptake in these cells.
Data represent mean values of fluorescence intensity ± standard derivation (n=30).
Three replicates were carried out. Representative results are shown. FSC: forward-scattered light; SSC: side-scattered light. Fluorescence signals were collected at 530 ± 30 nm upon excitation at 488 nm.
a) Raw 264.7 macrophages were stained with BClO only. b) Raw 264.7 macrophages were treated with LPS (1 µg/mL) for 24 h and PMA (1 µg/mL) for 1 h, followed by the incubation with BClO (1 µM) for 20 min.14.
After the addition of elesclomol (2 µM) to MCF-7 cells that were prestained with BClO (1 µM), fluorescence images were recorded at different time points: a) 0 min; b) 30 min; c) 60 min; d) 90 min; e) 120 min; f) 180 min.14.
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