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  • Effects of different detachment procedures on viability, nitroxide reduction kinetics, and plasma membrane heterogeneity of V-79 cells
    Batista, Urška ...
    Cell detachment procedures can cause severe damage to cells. Many studies require cells to be detached before measurements; therefore research on cells that have been grown attached to the bottom of ... the culture dish and later detached represents a special problem with respect to the experimental resultswhen the properties of cell membranes undergo small changes like in spectroscopic studies of membrane permeability. We characterized the influence of three different detachment procedures: cell scraping by rubber policeman, trypsinization, and a citrate buffer treatment on V-79 cells in plateau phase of growth (arrested in G1). We have measured cell viability by dye exclusion test; nitroxide reduction kinetics and membrane fluidity by electron paramagnetic resonance (EPR) method using lipophilic spin-probe MeFASL (10,3), which partitions mainly in cell membranes and hydrophilic spin-probe TEMPONE. The resulting cell damage due to detachment process was observed with scanning electron microscopy (SEM). We found out that cell viability was: 91% for trypsin, 85% for citrate and 70% for cell scraping. Though the plasma membrane was mechanically damaged by scraping, the membrane domain structure was not significantly altered compared to other detachment methods. On the other hand the spin-probe reduction rate, which depends both, on the transport across plasma membrane as well as on metabolic properties of cells, was the highest for trypsin method, suggesting that metabolic rate was the least influenced. Only the reduction rate of trypsin treated cells stayed unchanged after four hours of stirring in suspension. These results suggest that, compared to scraping cells or using citrate buffer, the most suitable detachment method for V-79 cells is detachment by trypsin and keeping cells inthe stirred cell suspension until measurement. (Abstract truncated at 2000 characters)
    Source: Cell biology international. - ISSN 1065-6995 (Letn. 34, št. 6, 2010, str. 663-668)
    Type of material - article, component part
    Publish date - 2010
    Language - english
    COBISS.SI-ID - 26810841
    DOI

source: Cell biology international. - ISSN 1065-6995 (Letn. 34, št. 6, 2010, str. 663-668)
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