Monitoring of membrane phospholipid scrambling in human erythrocytes and K562 cells with FM1-43 - a comparison with annexin V-FITC.
The styryl dye FM1-43 becomes highly fluorescent upon binding to cell membranes. The breakdown of membrane phospholipid asymmetry in ionophore-stimulated T-lymphocytes further increases this fluorescence [Zweifach, 2000]. In this study, the capacity of FM1-43 to monitor membrane phospholipid scrambling was explored using flow cytometry in human erythrocytes and human erythrocyte progenitor K562 cells. The Ca(2+)-dependent phosphatidylserine-specific probe annexin V-FITC was used for comparison. The presented data show that the loss of phospholipid asymmetry that could be induced in human erythrocytes by elevated intracellular Ca(2+) or by structurally different membrane intercalated amphiphilic compounds increases the FM1-43 fluorescence two- to fivefold. The profile of FM1-43 fluorescence for various treatments resembles that of phosphatidylserine exposure reported by annexin V-FITC. FM1-43 detected the onset of scrambling more efficiently than annexin V-FITC. The amphiphile-induced scrambling was shown to be a Ca(2+)-independent process. Monitoring of scrambling in K562 cells caused by NEM-induced Ca(2+)-release from intracellular stores and by Ca(2+) and ionophore A23187 treatment showed that the increase in FM1-43 fluorescence correlated well with the number of annexin V-FITC-detected phosphatidylserine-positive cells. The results presented here show the usefulness of FM1-43 as a Ca(2+)-independent marker of dissipation in asymmetric membrane phospholipid distribution induced by various stimuli in both nucleated and non-nucleated cells.
Institute of Physics, Wrocław University of Technology, 50-370, Wrocław, Poland.
Anna
A
Wróbel
Małgorzata
M
Bobrowska-Hägerstrand
Christer
C
Lindqvist
Henry
H
Hägerstrand
D017304:Annexin A5 / Q000494:pharmacology*; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004910:Erythrocyte Membrane / Q000378:metabolism*; D004912:Erythrocytes / Q000187:drug effects* / Q000378:metabolism; D005033:Ethylmaleimide / Q000737:chemistry / Q000494:pharmacology; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives* / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000494:pharmacology; D006801:Humans; D020014:K562 Cells; D011726:Pyridinium Compounds / Q000494:pharmacology*; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology*
D017304:Annexin A5; D004791:Enzyme Inhibitors; C492760:FITC-annexin A5; C073804:FM1 43; D005456:Fluorescent Dyes; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; D000001:Calcimycin; D016650:Fluorescein-5-isothiocyanate; D005033:Ethylmaleimide; D002118:Calcium