The use of sequential staining for detection of heterogeneous intracellular response of individual Jurkat cells to lysophosphatidylcholine.
Living cells are known to exhibit great morphological, functional, spatial and temporal heterogeneity. Hence, the study of cells in a bulk, whether this bulk is homogenous or heterogeneous, does not provide sufficiently detailed or interpretable results. An advantageous approach would rather be a comprehensive study of cell biological activity in single isolated living cells. In this study, we present an imaging approach for studying pre-apoptotic and very early apoptotic events, during cell death induced by lysophosphatidylcholine (LPC) at the single cell level. The aim of this study is to investigate intracellular events, such as the mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) formation, before and immediately after LPC introduction to the lymphocytes at the level of individual cells. A new protocol of sequential staining was developed to study the relation between early apoptosis signs (PS externalization), MMP changes and intracellular ROS production rates at an individual Jurkat cell resolution. Simultaneous kinetic assessments of MMP, intracellular ROS levels and phosphatidylserine (PS) externalization were performed at a single cell resolution, using Optical LiveCell™ Array technology and image analysis. The parameters were measured and analyzed both before and during exposure to inducers in a Jurkat cell population, including three groups of single cells: spontaneous apoptotic cells, induced apoptotic cells and fully functional living cells. Exogenous LPC caused a heterogeneous intracellular response among Jurkat cells immediately after its introduction. Subgroups of cells with opposite changes of MMP and different kinetics of ROS increase, were revealed within the whole cell population. The subset of apoptosis-induced Jurkat cells, which became apoptotic within 3h after the LPC introduction, exhibited higher initial MMP compared to fully functional or spontaneous apoptotic cells. LPC-induced apoptosis was accompanied by a concomitant increase in intracellular ROS levels. In the present study, a method is described to assess the intracellular events in cells which were initially different in their physiological status. The individual T lymphocytes (Jurkat cells) in vitro have various susceptibilities to LPC effects at the very early stage of contact with the inducer. The apoptotic effect of LPC in individual Jurkat cells is associated with a relatively higher initial MMP before the introduction of the inducer and with a faster ROS formation within the affected cells. Such divergence may be significant in regulating the balance of lymphocyte subsets in pathological sites, either maintaining or preventing the inflammation components of atherosclerosis. We conclude that the presented approach provides the researcher, not only with the cell retaining methodology, but with opportunities to observe and find the distinctive cell subsets within the whole cell population as well, thus helping to define more exactly the role and importance of such sub-populations in physiological or pathological conditions.
The Biophysical Interdisciplinary Schottenstein Center for the Research and Technology of the Cellome, Bar-Ilan University, Ramat Gan 52900, Israel. afrimzon@yahoo.com
Elena
E
Afrimzon
Naomi
N
Zurgil
Yana
Y
Shafran
Pnina
P
Leibovich
Maria
M
Sobolev
Larissa
L
Guejes
Mordechai
M
Deutsch
D017209:Apoptosis / Q000187:drug effects*; D006801:Humans; D042541:Intracellular Space / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism; D019169:Jurkat Cells; D007700:Kinetics; D015458:Leukemia, T-Cell / Q000378:metabolism / Q000473:pathology; D008244:Lysophosphatidylcholines / Q000494:pharmacology*; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008855:Microscopy, Electron, Scanning; D008856:Microscopy, Fluorescence; D018715:Microscopy, Video; D017382:Reactive Oxygen Species / Q000378:metabolism; D015203:Reproducibility of Results; D012235:Rhodamines / Q000737:chemistry / Q000378:metabolism; D059010:Single-Cell Analysis / Q000379:methods*; D013194:Staining and Labeling / Q000379:methods*; D013997:Time Factors
D008244:Lysophosphatidylcholines; D017382:Reactive Oxygen Species; D012235:Rhodamines; C401833:tetramethylrhodamine methyl ester