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Flow cytometric determination of PMCA‐mediated Ca 2+ ‐extrusion in individual red blood cells
Author(s) -
de Jong Kitty,
Kuypers Frans A.
Publication year - 2007
Publication title -
cytometry part a
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.316
H-Index - 90
eISSN - 1552-4930
pISSN - 1552-4922
DOI - 10.1002/cyto.a.20429
Subject(s) - plasma membrane ca2+ atpase , flow cytometry , population , red blood cell , biology , microbiology and biotechnology , atpase , cell , cytosol , biochemistry , enzyme , demography , sociology
Background: Differences among red blood cells in the activity of the plasma membrane Ca 2+ ‐ATPase (PMCA) can impact cell signaling and survival. However, no method has been reported that measures this activity directly in individual cells. Methods: We have designed a novel assay for PMCA activity that uses the fluorescent Ca 2+ ‐reporter Fluo4 and flow cytometric analysis. The method recognizes the extrusion of Ca 2+ from the cell after a short Ca 2+ ‐loading pulse, which avoids the problem of ATP depletion and ascertains activity at V max capacity. Results: Our assay is responsive to known PMCA inhibitors, and while not intended for quantitative kinetic analysis of Ca 2+ ‐pumping, it can be used to determine qualitative differences between red blood cell populations that vary in PMCA activity. Using this assay, we confirmed that a normal red blood cell population shows heterogeneity with respect to the PMCA V max . Conclusion: We report a novel assay of PMCA activity in red blood cells that can provide qualitative information on PMCA activity in individual cells. © 2007 International Society for Analytical Cytology