1,721,041 research outputs found
CD34 SUBSETTING: A TOOL TO EVALUATE THE RECONSITUTIVE POTENTIAL OF HAEMATOPOIETIC TRANPLANTATION UNITS
No full text
DIFFERENTIAL USE OF GRANULE RELEASE, FAS LIGAND AND TRAIL CYTOTOXIC MECHANISM BY MATURE AND IMMATURE NK CELLS
No full textSupravital exposure to propidium iodide identifies apoptotic cells in the absenceof nucleosomal DNA fragmentation
No full textBy flow cytometry, we have quantitatively evaluated HL-60, MOLT-4, and P815
apoptotic cells induced with camptothecin, staurosporine, and hyperthermia,
respectively. Apoptosis was measured by two different flow cytometry techniques,
using propidium iodide (PI) uptake in permeabilized and nonpermeabilized cells.
Apoptosis also has been analyzed by electron microscopy and DNA cleavage by in
situ nick translation and DNA gel electrophoresis. We demonstrate that supravital
exposure to propidium iodide without prior permeabilization identifies apoptotic
cells, and clearly distinguishes them from necrotic cells in all the cases
examined. This capability is independent of the nucleosomal (180-200 bp)
fragmentation of DNA (which does not take place in MOLT-4 cells), which is the
basis for detection of apoptosis both as a "ladder" by DNA gel electrophoresis
and as a hypodiploid peak by flow cytometry. Therefore, alterations in membrane
permeability, on which PI uptake in living cells is based, allow distinction of
apoptotic cells from necrotic and living cells independently of the heterogeneous
biochemical patterns involved in programmed cell death, which may or may not lead
to DNA oligonucleosomal fragmentation
Subtraction of autofluorescent dead cells from the lymphocyte flow cytometric binding assay.
No full textFlow cytometry allows the quantitative analysis of lymphocyte-target cell
conjugates and the identification of the lymphocyte subset involved in the
binding phenomenon. We recently described a methodology to identify the effector
cells bound to K562 targets based on target cell autofluorescence coupled with
lymphocyte staining by means of fluorescent monoclonal antibodies. Here we
describe an implementation of the methodology that allows the subtraction of
spontaneously dead targets to which lymphocytes may or may not adhere, thereby
preventing the overestimation of the binding phenomenon and limiting its
evaluation to living effector-target conjugates, thus preserving the specificity
of the phenomenon
Optimal detection of apoptosis by flow cytometry depends on cell morphology.
No full textFlow cytometry has recently become a choice technique for the quantitative
analysis of apoptosis. Monoparametric DNA analysis usually allows identification
of apoptotic cells as a "subdiploid" peak. Progression through apoptosis leads to
chromatin condensation, nuclear fragmentation and eventually to cell disruption.
Thus, a major problem for the flow cytometric analysis of apoptotic populations
is discrimination between debris and apoptotic cells. Here we demonstrate that
the best parameter on which to make such a distinction is the DNA content, no
matter what type of cell is studied. In contrast, discrimination between
apoptotic, non-apoptotic cells, and debris is possible on the basis of scattering
signals only in few selected cases, depending on the morphology of the intact
cells
Assays of natural killer (NK) cell ligation to target cells
No full textAssays for natural killer cells have long been established in the flow
repertoire, but successful application of a flow-based protocol requires
attention to many details. This unit provides these details in a comprehensive
manner. In particular, the assay is designed for simple bench-top cytometers,
rather than for more complex research instruments. Following this protocol, even
a novice user should be able to achieve successful completion of an NK assay
Differential kinetics of propidium iodide uptake in apoptotic and necrotic thymocytes.
No full textApoptosis and necrosis represent two different mechanisms by which cells die. The
dynamics of cellular lesions in these two processes differ. In particular we
demonstrate that plasma membrane damage, occurring as a primary event during
necrosis represents, on the contrary, a delayed but massive phenomenon during
apoptosis. In consequence there are different kinetics of propidium iodide
incorporation by necrotic and apoptotic thymocytes. This represents the basis for
the flow cytometric identification of different cellular subsets. Analysis of
these subsets after sorting showed that clearly apoptotic cells, which are not
able to exclude propidium iodide for long incubation periods, do not show any
morphologically detectable membrane damage. The kinetics of propidium iodide
incorporation in vivo in isolated rat thymocytes can therefore be used in flow
cytometric analysis. This technique can be used instead of DNA staining of
ethanol-treated cells or nick translation to recognize apoptotic cells, and
distinguish apoptosis from necrosis, without killing the cell
- …
