Document Detail

Effects of different detachment procedures on viability, nitroxide reduction kinetics and plasma membrane heterogeneity of V-79 cells.
MedLine Citation:
PMID:  20337597     Owner:  NLM     Status:  MEDLINE    
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 results when the properties of cell membranes undergo small changes such as 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 the plateau phase of growth (arrested in G1). We have measured cell viability by a dye-exclusion test; nitroxide reduction kinetics and membrane fluidity by EPR (electron paramagnetic resonance) method using the lipophilic spin-probe MeFASL(10,3) (5-doxylpalmitoyl-methylester), which partitions mainly in cell membranes and the hydrophilic spin-probe TEMPONE (4-oxo-2,2,6,6-tetramethylpiperidine-1-oxyl). The resulting cell damage due to the detachment process was observed with SEM (scanning electron microscopy). We found out that cell viability was 91% for trypsin treatment, 85% for citrate treatment and 70% for cell scraping. Though the plasma membrane was mechanically damaged by scraping, the membrane domain structure was not significantly altered compared with 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 4 h of stirring in suspension. These results suggest that, compared with scraping cells or using citrate buffer, the most suitable detachment method for V-79 cells is detachment by trypsin and keeping cells in the stirred cell suspension until measurement. This method provides the highest cell viability, less visible damage on SEM micrographs and leaves the metabolic rate of cells unchanged.
Urska Batista; Maja Garvas; Marjana Nemec; Milan Schara; Peter Veranic; Tilen Koklic
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2010-05-07
Journal Detail:
Title:  Cell biology international     Volume:  34     ISSN:  1095-8355     ISO Abbreviation:  Cell Biol. Int.     Publication Date:  2010 Jun 
Date Detail:
Created Date:  2010-05-10     Completed Date:  2010-07-28     Revised Date:  2010-11-12    
Medline Journal Info:
Nlm Unique ID:  9307129     Medline TA:  Cell Biol Int     Country:  England    
Other Details:
Languages:  eng     Pagination:  663-8     Citation Subset:  IM    
Institute of Biophysics, Faculty of Medicine, University of Ljubljana, 1000 Ljubljana, Slovenia.
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MeSH Terms
Cell Culture Techniques*
Cell Line
Cell Membrane / metabolism,  physiology
Cell Membrane Permeability*
Cell Survival
Citrates / pharmacology
Cyclic N-Oxides / chemistry
Electron Spin Resonance Spectroscopy*
Membrane Fluidity*
Microscopy, Electron, Scanning
Nitrogen Oxides / metabolism*
Spin Labels
Stress, Mechanical
Triacetoneamine-N-Oxyl / chemistry
Trypsin / pharmacology
Reg. No./Substance:
0/5-doxylpalmitoyl-3-phosphatidylcholine; 0/Citrates; 0/Cyclic N-Oxides; 0/Nitrogen Oxides; 0/Spin Labels; 2896-70-0/Triacetoneamine-N-Oxyl; EC

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