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Abstract <p>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&#xa0;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 <i>ex&#xa0;vivo</i> and also enable the use of multicolor FACS analysis and sorting of live LacZ-positive leukocytes for further genetic and functional studies.</p>

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

  • 期刊论文

  • 发表日期：

    2025年5月1日

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  • Veronika S. Mihailovskaya,
  • Daria A. Bogdanova,
  • Oleg N. Demidov,
  • Stanislav A. Rybtsov

摘要

Abstract

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.