Flow cytometric determination of PMCA-mediated Ca2+-extrusion in individual red blood cells

✍ Scribed by Kitty de Jong; Frans A. Kuypers

• Publisher: John Wiley and Sons
• Year: 2007
• Tongue: English
• Weight: 751 KB
• Volume: 71A
• Category: Article
• ISSN: 0196-4763
• DOI: 10.1002/cyto.a.20429

✦ Synopsis

Abstract

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