Abstract
Phosphatidylserine (PS) exposure occurs during the cell death program and fluorescein-labeled lactadherin permits the detection of PS exposure earlier than annexin V in suspended cell lines. Adherent cell lines were studied for this apoptosis-associated phenomenon to determine if PS probing methods are reliable because specific membrane damage may occur during harvesting. Apoptosis was induced in the human tongue squamous carcinoma cell line (Tca8113) and the adenoid cystic carcinoma cell line (ACC-2) by arsenic trioxide. Cells were harvested with a modified procedure and labeled with lactadherin and/or annexin V. PS exposure was localized by confocal microscopy and apoptosis was quantified by flow cytometry. The detachment procedure without trypsinization did not induce cell damage. In competition binding experiments, phospholipid vesicles competed for more than 95 and 90% of lactadherin but only about 75 and 70% of annexin V binding to Tca8113 and ACC-2 cells. These data indicate that PS exposure occurs in three stages during the cell death program and that fluorescein-labeled lactadherin permitted the detection of early PS exposure. A similar pattern of PS exposure has been observed in two malignant cell lines with different adherence, suggesting that this pattern of PS exposure is common in adherent cells. Both lactadherin and annexin V could be used in adherent Tca8113 and ACC-2 cell lines when an appropriate harvesting procedure was used. Lactadherin is more sensitive than annexin V for the detection of PS exposure as the physical structure of PS in these blebs and condensed apoptotic cell surface may be more conducive to binding lactadherin than annexin V.
Highlights
Sequential changes occur in the plasma membrane during apoptosis
This approach circumvents the problem of measuring falsepositive cells due to membrane damage inflicted upon healthy cells during their detachment from the culture flasks. Both lactadherin and annexin V can be used in the study of PS exposure on the surface of adherent cells during apoptosis
Flow cytometry analysis indicated that untreated Tca8113 and ACC-2 exhibited little staining with lactadherin/annexin V or Propidium iodide (PI), indicating that the detachment procedure did not induce cell damage
Summary
The asymmetry in the distribution of the phospholipids is impaired and phosphatidylserine (PS), which normally is located on the internal leaflet of the membrane, is exposed on the cell surface This event can be detected by acquisition of the ability of the cell to bind anticoagulant proteins such as lactadherin and annexin V [1,2,3]. To determine whether lactadherin or annexin V is more appropriate for the detection of apoptosis in adherent cell populations, a previously described modified procedure [5] was used to harvest cells This approach circumvents the problem of measuring falsepositive cells due to membrane damage inflicted upon healthy cells during their detachment from the culture flasks. Both lactadherin and annexin V can be used in the study of PS exposure on the surface of adherent cells during apoptosis
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