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Journal ArticleDOI

Difference in Viability of CD34+ Cells in Cryopreserved Cord Blood According to Evaluation Methods

01 Jun 2009-The Korean Journal of Hematology (The Korean Society of Hematology)-Vol. 44, Iss: 2, pp 92-99
TL;DR: After thawing the cryopreserved UCB, 89% of the total MNCs and 84% of CD34+ cells were viable as identified by trypan blue exclusion assay, and the cell death rate was found to be 47% by Annexin-V/PI staining and less than 5% by DNA contents analysis.
Abstract: Background: On performing umbilical cord blood (UCB) transplantation, faster engraftment may lead better clinical outcome. Because transplanted viable cell count in UCB is related to the engraftment, accu- rate evaluation of viability of CD34+ cells in cryopreserved UCB has clinical implication. We examined the difference in viability of cells in cryopreserved UCB according to the duration of cryopreservation and different methods. Methods: A total of 60 UCB samples which were cryopreserved for 1 to 4 years were used in this study. Viability of cryopreserved cells were examined with trypan blue exclusion assay, DNA contents analysis, caspase-3 activation test, intracellular esterase activity and Annexin-V/PI staining. Results: After thawing the cryopreserved UCB, 89% of the total MNCs and 84% of CD34+ cells were viable as identified by trypan blue exclusion assay. In the CD34+ cell population, the cell death rate was found to be 47% by Annexin-V/PI staining and less than 5% by DNA contents analysis. However, cspase-3 activity failed to document apoptosis. The intracellular esterase activity test also showed a cell death rate of about 10 ∼20% at 2, 4, and 6 hours after thawing.

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Book ChapterDOI
TL;DR: This chapter briefly describes the biochemical basis for each of these techniques for detecting PCD, and then attempts to summarize the strengths and weaknesses of each approach.
Abstract: Publisher Summary The growing awareness of the frequency and importance of physiological cell death (PCD) has resulted from two related developments, an increasing understanding of the biochemistry of PCD and a utilization of that understanding to develop sensitive methods for detecting PCD. The realization that this form of cell death is generally accompanied by internucleosomal DNA cleavage led to a variety of techniques for detecting this process, including DNA electrophoresis, flow cytometry, filtration assays, and in situ nick end labeling. Recent studies have demonstrated that PCD is accompanied by loss of phospholipid asymmetry across the plasma membrane as well as activation of various proteases, particularly members of the interleukin-1/3 converting enzyme (ICE) family. This chapter briefly describes the biochemical basis for each of these techniques and then attempts to summarize the strengths and weaknesses of each approach. In this regard Inhibitors of proteases (and other enzymes) are potentially useful tools when employed properly. Because the selectivity of these inhibitors is not fully known, however, experiments must be carefully designed, suitably controlled, and cautiously interpreted. Moreover, multiple alternative techniques (including antisense approaches and genetic knockouts) must be employed to confirm or refute conclusions suggested by inhibitor studies.

57 citations

Journal ArticleDOI
TL;DR: The ML-IC assay demonstrates that the greater repopulating ability of UCB is due to the higher generative ability of HSC in UCB, and can discriminate between cytokine-mediated expansion of hematopoietic progenitors by enhancing generation of immature daughter cells or by recruiting otherwise quiescent cells.

51 citations

Journal ArticleDOI
TL;DR: It is found that even after cryopreservation the sequestering of endogenous phosphatidylserine to the cytoplasmic leaflet is maintained in intact cells, but not in impaired cells, and the physiological consequences of a perturbed transbilayer asymmetry in sperm plasma membranes is discussed.
Abstract: The transbilayer dynamics of lipids in the plasma membrane of mammalian sperm cells is crucial for the fertilization process. Here, the transbilayer movement and distribution of phospholipids in the plasma membrane of fresh, ejaculated and cryopreserved ram spermatozoa was studied by labeling cells with fluorescent analogues of phosphatidylserine and phosphatidylcholine. By co-labeling cells with the DNA-binding dye propidiumiodide as well as by employing fluorescence microscopy and flow cytometry we were able to determine the transbilayer redistribution of fluorescent phospholipid analogues in intact (propidiumiodide-negative) and in impaired (propidiumiodide-positive) spermatozoa. The transbilayer distribution of the fluorescent phosphatidylserine and phosphatidylcholine analogues was not perturbed in intact sperm cells after cryopreservation. In those cells, the phosphatidylserine analogue became rapidly enriched on the cytoplasmic leaflet by the activity of a putative aminophospholipid translocase similar to intact cells of fresh, ejaculated samples. However, upon cryopreservation the activity of the putative aminophospholipid translocase was significantly reduced in intact cells. Employing annexin V-FITC, we found that even after cryopreservation the sequestering of endogenous phosphatidylserine to the cytoplasmic leaflet is maintained in intact cells, but not in impaired cells. The phosphatidylcholine analogue redistributed very slowly remaining essentially confined to the exoplasmic leaflet of the plasma membrane of intact cells from both fresh, ejaculated and cryopreserved samples. The physiological consequences of a perturbed transbilayer asymmetry in sperm plasma membranes is discussed.

50 citations

Journal ArticleDOI
TL;DR: This work attempts to provide further insights into mechanisms of reduced SYTO16 fluorescence during apoptosis, and identifies three mechanisms that may be responsible for this phenomenon.
Abstract: Background: SYTO probes are gaining momentum as reliable and easy to use markers of apoptotic cell death, but the phenomenon underlying reduced SYTO fluorescence in apoptotic cells as compared with normal cells is still not fully elucidated. Herein, we attempt to provide further insights into mechanisms of reduced SYTO16 fluorescence during apoptosis. Methods: Human follicular lymphoma cell lines were subjected to diverse apoptotic and oncotic stimuli with subsequent multiparametric flow cytometric and fluorescence imaging analysis. SYTO green (SYTO11-16), TMRM, PI, 7AAD, and Hoechst 33342 probes were applied for multivariate analysis of temporal sequence of apoptotic events. Sorting of cells differing in the level of SYTO16 fluorescence and subsequent characterization of obtained subpopulations were also performed. Results: Loss of SYTO16 fluorescence (SYTOlow/PI+ events) has been observed in cells exposed to oncotic stimuli, whereas SYTOhigh/PI+ events did not prevail at any treatment scenario. We tracked similarities and discrepancies between SYTO16 and TMRM probes. Often, SYTO16 and TMRM exhibited the same staining profiles, as loss of their fluorescence was detected in a single cell population. However, both mitochondrial uncoupler FCCP and a small-molecule Bcl-2 inhibitor, HA14-1, appeared to induce distinct staining profiles of SYTO16 and TMRM, with the decrease in TMRM fluorescence preceding the loss of SYTO16 fluorescence. Importantly, in both cases (FCCP and HA14-1) the decrease of SYTO16 fluorescence was blocked by pharmacological inhibition of caspases (with z-VAD-fmk). Conclusions: The data demonstrate that loss of SYTO16 is caspase-dependent, as is not a mere indicator of Δψm dissipation. Commonly observed similarities between SYTO and TMRM may stem from the fast kinetics of apoptotic events once cell death is initiated. © 2007 International Society for Analytical Cytology.

37 citations

Journal ArticleDOI
TL;DR: Early apoptosis of CD34+ cells could influence the outcome of transplantation using cryopreserved UCB, and the Annexin V assay identified most repopulating activities in UCB units.
Abstract: The increased use of umbilical cord blood (UCB) raises issues regarding the quality of cryopreserved UCB This study investigated whether early apoptosis of CD34+ cells is part of the functional heterogeneity of cryopreserved UCB Annexin V binding of CD34+ PI(-) cells showed wide variations in both fresh and cryopreserved UCBs, with greater variation among units frozen for > 5 years Xenotransplantation of sorted cells into non-obese diabetic severe combined immunodeficient mice demonstrated that the Annexin V assay identified most repopulating activities in UCB units Thus, early apoptosis of CD34+ cells could influence the outcome of transplantation using cryopreserved UCB

26 citations

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