(Authors): Institute of Pathology, University of Regensburg
Background: Over the past decade cell separation has become an important tool in cell biology not only for direct analysis of defined cell fractions but also for 're'-cultivation of cell subpopulations.
Objective: The aim of our study was to investigate if cell separation by FACS (FACStarPLUS, BD) and/or MACS (Miltenyi Biotec) is associated with changes in parameters reflecting cell membrane physiology such as membrane potential, viscosity, and asymmetry.
Materials and Methods: Cell separation was carried out on BT474 breast carcinoma cells and N1 skin fibroblasts using a fibroblast-specific marker (AS02, Dianova) and a magnetic-bead-conjugated secondary antibody (Miltenyi Biotec) for MACS. Membrane potential was determined with the fluorescence indicator DiBAC4 (3) (Radosevic et al., J. Immunol. Methods 161:119,1993). A monomer/excimer method utilizing PDA (Galla & Sackman, Biochim. Biophys. Acta 339, 103, 1974) was used to investigate membrane viscosity and membrane asymmetry was measured via Annexin-V-FITC staining of phosphatidylserine in the outer leaflet of the plasma membrane (Van Engeland et al., Cytometry 24, 131, 1996). Propidiumiodide-positive cells were excluded from further calculations.
Results: In general, neither MACS nor FACS influenced cell membrane viscosity. In contrast, a clear increase in cell membrane potential was registered following separation via MACS for both cell lines and for N1 cells after FACS. In addition, cell sorting of BT474 tumor cells by both techniques resulted in the generation of an Annexin-V-positive, PI-negative cell population of 20-25% of the total cell count whereas N1 fibroblasts were unaffected.
(Supported by: DFG grants Ku 971/2-1 and /2-2, Miltenyi Biotec Inc., and Dianova GmbH.)