title: Phosphatidylserine Exposure in Human Red Blood Cells Depending on Cell Age.
abstract: OBJECTIVE
  The exposure of phosphatidylserine (PS) on the outer membrane leaflet of red blood cells (RBCs) serves as a signal for suicidal erythrocyte death or eryptosis, which may be of importance for cell clearance from blood circulation. PS externalisation is realised by the scramblase activated by an increase of intracellular Ca2+ content. It has been described in literature that RBCs show an increased intracellular Ca2+ content as well as PS exposure when becoming aged up to 120 days (which is their life span). However, these investigations were carried out after incubation of the RBCs for 48 h. The aim of this study was to investigate this effect after short-time incubation using a variety of stimulating substances for Ca2+ uptake and PS exposure.
  
  
  METHODS
  We separated RBCs by age in five different fractions by centrifugation using Percoll density gradient. The intracellular Ca2+ content and the PS exposure of RBCs with different age has been investigated after treatment with lysophosphatidic acid (LPA) as well as after activation of protein kinase C (PKC) using phorbol-12 myristate-13 acetate (PMA). For positive control RBCs were treated with 4-bromo-A23187. Measurement techniques included flow cytometry and live cell imaging (fluorescence microscopy).
  
  
  RESULTS
  The percentage of RBCs showing increased Ca2+ content as well as the PS exposure did not change significantly in dependence on cell age after short-time incubation in control experiments (without stimulating substances) or using LPA or PMA. However, we confirm findings reported that Ca2+ content and the PS exposure of RBCs increased after 48 h incubation.
  
  
  CONCLUSIONS
  No significant differences of intracellular Ca2+ content and PS exposure can be seen for RBCs of different age in resting state or after stimulation of Ca2+ uptake at short-time incubation.
authors.forename: Mauro C
authors.initials: MC
authors.lastname: Wesseling
authors.forename: Lisa
authors.initials: L
authors.lastname: Wagner-Britz
authors.forename: Henri
authors.initials: H
authors.lastname: Huppert
authors.forename: Benjamin
authors.initials: B
authors.lastname: Hanf
authors.forename: Laura
authors.initials: L
authors.lastname: Hertz
authors.forename: Duc Bach
authors.initials: DB
authors.lastname: Nguyen
authors.forename: Ingolf
authors.initials: I
authors.lastname: Bernhardt
mesh_terms: D000367:Age Factors; D002118:Calcium / Q000378:metabolism; D048429:Cell Size / Q000187:drug effects; D002478:Cells, Cultured; D004912:Erythrocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D008246:Lysophospholipids / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D010718:Phosphatidylserines / Q000494:pharmacology*; D011493:Protein Kinase C / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D008246:Lysophospholipids; D010718:Phosphatidylserines; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; C032881:lysophosphatidic acid; D002118:Calcium