Multiplex Immune Profiling of Mouse Blood Cells with Highly Sensitive Detection of β-Galactosidase LacZ Reporter

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Veronika S. Mihailovskaya¹, Daria A. Bogdanova^1,2, Oleg N. Demidov^1,2, Stanislav A. Rybtsov^1,a*

¹Division of Immunobiology and Biomedicine, Scientific Center for Genetics and Life Sciences, , Sirius University of Science and Technology, 354340 Sirius, Russia

²Institute of Cytology, Russian Academy of Sciences, 194064 St. Petersburg, Russia

^* To whom correspondence should be addressed.

Received: March 19, 2025; Revised: May 11, 2025; Accepted: May 14, 2025

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Bacterial β-galactosidase (LacZ) has been widely used as a reporter for the development of mice models to study gene expression or for control of conditional gene deletion. However, high level of endogenous β-galactosidase expression, and low sensitivity of existing substrates for detection of transgenic LacZ activity limited this reporter application for live cell analysis. To overcome this limitation, we evaluated performance of the intracellularly immobilizable fluorescent probe SPiDER-βGal to detect LacZ in major blood cell populations of the reporter mice using multicolor flow cytometry. SPiDER-βGal allows highly sensitive detection of LacZ but also detects endogenous β-galactosidase. Therefore, high background expression of the endogenous β-galactosidase in the myeloid cells impeded advantages of the SPiDER-βGal probe. Application of the proton pump inhibitor – Bafilomycin A1 elevates lysosomal pH and increased specificity of the LacZ detection in the leukocyte populations by suppressing background endogenous β-galactosidase activity. Extending incubation with SPiDER-βGal to 60 minutes also increased sensitivity of the assay tenfold. Thus, lysosomal proton transport inhibitors increase resolution of the LacZ analysis in the cells of reporter animals with high endogenous β-galactosidase activity ex vivo and also enable the use of multicolor FACS analysis and sorting of live LacZ-positive leukocytes for further genetic and functional studies.

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KEY WORDS: multicolor flow cytometry, Bafilomycin A1, β-galactosidase, LacZ, populations of blood cells, leukocytes, reporter mice

DOI: 10.1134/S0006297925600796

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