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Expression and visualization during cell cycle progression of alkaline phosphatase in B lymphocytes from C3H/HeJ mice
V Souvannavong1, S Brown, M Sarih
1CNRS-URA 1116, Institut de Biochimie, Université Paris-Sud, Orsay, France.
Insights
Alkaline phosphatase (APase) activity in B cells from C3H/HeJ mice is optimally induced by interleukin-5 and dextran sulfate (DXS), not lipopolysaccharide (LPS). APase expression and localization vary with cell cycle phase, particularly in S and G2/M stages.
Area of Science:
- Immunology
- Cell Biology
Background:
- Lipopolysaccharide (LPS) typically induces alkaline phosphatase (APase) in B lymphocytes.
- C3H/HeJ mice exhibit hyporesponsiveness to LPS stimulation.
Purpose of the Study:
- To investigate alkaline phosphatase (APase) expression in B cells from LPS-hyporesponsive C3H/HeJ mice.
- To determine the effect of different stimuli on APase activity and its cell cycle-dependent localization.
Main Methods:
- Purification of B cells from C3H/HeJ mice.
- Stimulation with interleukin-5 (IL-5), dextran sulfate (DXS), and LPS.
- Flow cytometry for simultaneous APase activity and DNA content analysis.
- Confocal microscopy for visualizing APase localization in sorted cells.
Main Results:
- Optimal APase activity was achieved with IL-5 and DXS, while LPS failed to induce expression.
- APase was highly expressed in G1 phase cells and in over 50% of cells in S and G2/M phases.
- APase localization shifted from cytoplasmic vesicles in G1 to peripheral patches in S and distinct capping in G2/M cells.
Conclusions:
- B cells from C3H/HeJ mice require specific costimulatory signals (IL-5 and DXS) for APase induction.
- APase expression and subcellular localization are tightly regulated by the cell cycle in these B cells.
Abstract:
The expression of alkaline phosphatase (APase) activity by purified B cells from lipopolysaccharide (LPS)-hyporesponsive C3H/HeJ mice was determined. Optimal APase activity was expressed after costimulation with interleukin-5 and dextran sulfate (DXS), whereas LPS, which is highly effective on B lymphocytes from normal mice, was unable to induce enzyme expression, even in the presence of DXS. The simultaneous determination by flow cytometry of both cellular APase, by using a fluorescent azo dye technique, and DNA content showed that APase was highly expressed by about one-tenth of cells in G1 phase, whereas it was present in more than 50% of cells in S and G2/M phases. The enzyme, as visualized by confocal microscopy after cell sorting on the basis of DNA content, was found to be localized mainly in vesicular structures distributed throughout the cytoplasm in G1 cells. It was distributed in patches and essentially localized at the cell periphery in S cells, whereas clear capping of activity was observed in G2/M cells.
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