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Monitoring of membrane phospholipid scrambling in human erythrocytes and K562 cells with FM1-43 — a comparison with annexin V-FITC

Abstract

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 Ca2+-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 Ca2+ 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 Ca2+-independent process. Monitoring of scrambling in K562 cells caused by NEM-induced Ca2+-release from intracellular stores and by Ca2+ 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 Ca2+-independent marker of dissipation in asymmetric membrane phospholipid distribution induced by various stimuli in both nucleated and non-nucleated cells.

Abbreviations

A23187:

calcium ionophore

BSA:

bovine serum albumin

C10E8:

octaethylene glycol mono n-decyl ether

C12E8:

octaethylene glycol mono n-dodecyl ether

C12T:

dodecyltrimethylammonium bromide

C12Z:

3-(dodecyldimethylammonio)-1-propanesulphonate

CPZ:

chlorpromazine hydrochloride

DMSO:

dimethyl sulfoxide

FITC:

fluorescein isothiocyanate

Fluo3-AM:

4-(6-acetoxymethoxy-2, 7-dichloro-3-oxo-9-xanthenyl)-4′-methyl-2,2′(ethylenedioxy)dianiline-N,N,N′,N′-tetraacetic acid tetrakis (acetoxymethyl) ester

FM1-43:

(n-(3-triethylammoniumpropyl)-4-(4-(dibutylamino) styryl) pyridinium dibromide)

IONO:

ionomycin

NEM:

n-ethylmaleimide

PMA:

phorbol 12-myristate 13-acetate

PS:

phosphatidylserine

S12S:

sodium dodecyl sulphate

S14S:

sodium tetradecyl sulphate

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Correspondence to Henry Hägerstrand.

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Wróbel, A., Bobrowska-Hägerstrand, M., Lindqvist, C. et al. Monitoring of membrane phospholipid scrambling in human erythrocytes and K562 cells with FM1-43 — a comparison with annexin V-FITC. Cell Mol Biol Lett 19, 262–276 (2014). https://doi.org/10.2478/s11658-014-0195-3

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Keywords

  • FM1-43
  • Phospholipid scrambling
  • Annexin V-FITC
  • Phosphatidylserine exposure
  • Human erythrocytes
  • K562 cells
  • Ionophores
  • Amphiphiles