Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “single cell analysis”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 163 records · Page 9Linked to original sources
PubMed2005

Real time single cell analysis of Bid cleavage and Bid translocation during caspase-dependent and neuronal caspase-independent apoptosis.

Bcl-2 homology domain (BH) 3-only proteins couple stress signals to evolutionarily conserved mitochondrial apoptotic pathways. Caspase 8-mediated cleavage of the BH3-only protein Bid into a truncated protein (tBid) and subsequent translocation of tBid to mitochondria has been implicated in death receptor signaling. We utilized a recombinant fluorescence resonance energy transfer (FRET) Bid probe to determine the kinetics of Bid cleavage and tBid translocation during death receptor-induced apoptosis in caspase 3-deficient MCF-7 cells. Cells treated with tumor necrosis factor-alpha (200 ng/ml) showed a rapid cleavage of the Bid-FRET probe occurring 75.4 +/- 12.6 min after onset of the tumor necrosis factor-alpha exposure. Cleavage of the Bid-FRET probe coincided with a translocation of tBid to the mitochondria and a collapse of the mitochondrial membrane potential (DeltaPsim). We next investigated the role of Bid cleavage in a model of caspase-independent, glutamate-induced excitotoxic apoptosis. Rat cerebellar granule neurons were transfected with the Bid-FRET probe and exposed to glutamate for 5 min. In contrast to death receptor-induced apoptosis, neurons showed a translocation of full-length Bid to the mitochondria. This translocation occurred 5.6 +/- 1.7 h after the termination of the glutamate exposure and was also paralleled with a collapse of the DeltaPsim. Proteolytic cleavage of the FRET probe also occurred, however, only 25.2 +/- 3.5 min after its translocation to the mitochondria. Subfractionation experiments confirmed a translocation of full-length Bid from the cytosolic to the mitochondrial fraction during excitotoxic apoptosis. Our data demonstrate that both tBid and full-length Bid have the capacity to translocate to mitochondria during apoptosis.

Animals↗
PubMed1993

Cytokine production in virus-infected mice: a single cell analysis.

Antiviral activity of IFN-gamma and TNF has been demonstrated in vitro and in vivo. Recombinant vaccinia viruses that encode either of these cytokines are highly attenuated in vivo and, indeed, are no longer lethal when they are used to infect athymic nude mice. Thus, these cytokines are able to replace the requirement for CD8 T cells in immunodeficient mice. We believe that the antiviral properties of some cytokines are a central component in the physiological control of virus infection. In this report, we used flow cytofluorimetric analysis to simultaneously detect cytokine production and T cell phenotype. Importantly, this method allowed the analysis of cytokine production in cells freshly isolated from tissues. In contrast to other procedures, restimulation of the cells in vitro was not necessary. We demonstrate that CD8 IFN-gamma+ cells were predominantly induced early in the response to vaccinia virus infection and, at the cellular peak of the response, CD8 IFN-gamma+ and CD4 TNF+ T cells were present at equal frequencies. Hence, these findings support the concept that effector T cells produce cytokines that are known to be antiviral, and that these cytokines are an important component of effector T cell activity.

Animals↗
PubMedDate not supplied

Detection of a feline X-linked antigen in somatic cell hybrids. Single-cell analysis using monoclonal antibodies.

We tested the premise that monoclonal antibodies to either intracellular or membrane antigens can greatly facilitate the construction of linkage maps of mammals whose chromosomes can be introduced into rodent cells. Monoclonal antibodies against antigenic determinants of cat lymphocytes and fibroblasts were used to analyze feline antigen expression in cat-mouse somatic cell hybrid populations selected to contain the X-linked feline HPRT locus. The frequency of antigen expression as measured by fixed cell immunofluorescence (IF) assays, varied greatly within hybrid populations for all but the antigen designated as VP382. Its frequent presence in hybrid cells led to the prediction, confirmed by 8-azaguanine selection experiments, that its expression was controlled by a gene, or genes, on the feline X chromosome. The antigens identified by the rest of the antibodies segregated independently of each other in cat-mouse somatic cell hybrids and their expression appeared to be controlled by autosomal genes of the cat.

Animals↗
PubMed1983

Growth and the inducibility of mycelium formation in Candida albicans: a single-cell analysis using a perfusion chamber.

In Candida albicans, cells actively growing in the budding form cannot be immediately induced to form a mycelium until they enter stationary phase. However, if exponential phase cells are starved for a minimum of 10 to 20 min, they are inducible. Using a video-monitored perfusion chamber, we found that starved cells were able to form mycelia regardless of their position in the budding cycle. When starved exponential cells were released into fresh nutrient medium at high temperature and pH, conditions conducive to mycelium formation, unbudded cells evaginated after an average lag period of 75 min and then grew exclusively in the mycelial form. Depending upon the volume, or maturity, of the bud, budded cells entered two different avenues of outgrowth leading to mycelium formation. If the daughter bud was small, growth resumed by apical elongation of the bud, leading to a 'shmoo' shape which tapered into an apical mycelium. If the daughter bud was large, the cell underwent a sequence of evaginations: first, the mother cell evaginated after an average period of 75 min; then the daughter bud evaginated 40 min later. Both evaginations then grew in the mycelial form. In this latter sequence, the evagination on the mother cell was positioned non-randomly, occurring in the majority of cells adjacent to the bud. All buds undergoing evagination contained a nucleus, but roughly 20% of buds undergoing apical elongation did not.

Candida albicans↗
PubMed1988

Single-cell analysis of the intracellular pH and its regulation during the monocytic differentiation of U937 human leukemic cells.

Monocytic differentiation of U937 cells induced by retinoic acid is accompanied by a 0.2-pH-unit cell alkalinisation. The effect of retinoic acid on intracellular pH (pHi) develops slowly and it precedes the differentiation of the cells by 24 h. Heterogeneity in cellular pHi values was assessed using flow cytometry. It was higher at the differentiated stage than at the undifferentiated stage. It was reduced under conditions of clamped pHi values. Two membrane mechanisms allow U937 cells to recover from an intracellular acidosis. These are the Na+/H+ exchange system and a Na+-dependent HCO3-/Cl- exchange system. The increase in the pHi observed after monocytic differentiation resulted from a twofold increase in the maximum activity of the Na+/H+ exchange system with no change in the activity of the bicarbonate-dependent system. The properties of interaction of the Na+/H+ exchanger of U937 cells with Na+, Li+, amiloride and its derivatives were defined and appeared to be unique to human leukemic cells.

Amiloride↗
PubMed1994

Muscular cavernous single cell analysis in patients with venoocclusive dysfunction.

Enzymatically isolated smooth muscle cells of the corpora cavernosa obtained from open biopsies of 15 patients, clinically nonresponding to papaverine/phentolamine and prostaglandin E1 (PGE1) and classified by cavernosometry, were examined using the patch-clamp technique in the whole-cell configuration mode simultaneously monitoring the intracellular calcium concentration by means of the Ca(2+)-sensitive fluorescence dye FURA-II. It could be demonstrated that extracellularly applied PGE1 induces smooth muscle relaxation by inhibition of voltage-dependent L-type Ca2+ currents (58 +/- 8%). Compared to intact cavenous tissue (n = 5), the smooth muscle cells of 14/15 PGE1 nonresponders had no evidence of functional disturbance. Due to intact smooth muscle cells in most cases, etiology of venoocclusive dysfunction remains unclear.

Alprostadil↗
PubMed1991

Single cell analysis of mesoderm formation in the Xenopus embryo.

We have examined the developmental specification of individual cells in the Xenopus blastula using a new in vitro culture system. Regional differences are apparent at the mid-blastula stage when animal hemisphere cells form only ectodermal cell types, while many clones from below the pigment boundary contain mesodermal cell types. A number of clones give rise to more than one differentiated cell type indicating that the initial steps of mesoderm induction are potentially reversible. Animal hemisphere cells can be induced to form mesoderm by fibroblast growth factor (FGF). Different cell types predominate at different FGF concentrations and the neighbours in this sequence are also the pairs of cell types most usually associated in mixed clones derived from the marginal zone. We propose that the specification of individual cells depends upon both the concentration of inducing factor and on stochastic intracellular events.

Animals↗
PubMed2003

Outer mitochondrial membrane permeabilization during apoptosis triggers caspase-independent mitochondrial and caspase-dependent plasma membrane potential depolarization: a single-cell analysis.

Little is known about the temporal relationship between mitochondrial and plasma membrane potential changes and outer mitochondrial membrane permeabilization during apoptosis. Confocal imaging of breast carcinoma and HeLa cells stably transfected with cytochrome-C-GFP demonstrated that mitochondria rapidly depolarized after the release of the fusion protein into the cytosol. Of note, mitochondria did not completely depolarize but established a new steady-state level that could be further dissipated by treatment with the protonophore carbonyl cyanide p-trifluoromethoxy-phenylhydrazone. Treatment with the F(O)F(1)-ATP-synthase inhibitor oligomycin likewise induced a collapse of this steady-state level, suggesting that F(O)F(1)-ATP-synthase reversal maintained mitochondrial potential after outer mitochondrial membrane permeabilization. Treatment with a broad spectrum caspase inhibitor failed to inhibit the partial depolarization of mitochondria during apoptosis, yet potently abolished the activation of effector caspases detected by fluorescence resonance energy transfer analysis in the same experiment. Interestingly, the onset of mitochondrial depolarization was always coupled with a depolarization of the plasma membrane potential. This was associated with the degradation of the regulatory Na(+)/K(+)-ATPase beta-subunit, and both events were blocked by caspase inhibition. Our results demonstrate that outer mitochondrial membrane permeabilization coordinates the depolarization of both membrane potentials during apoptosis.

Apoptosis↗
PubMed1989

DNA evaluation in growth hormone producing pituitary adenomas: flow cytometry versus single cell analysis.

DNA patterns were analysed in 26 GH-producing pituitary adenomas by flow cytometry as well as by microspectrophotometry. Twelve tumours (46%) were diploid according to both methods, whereas 5 tumours (19%) showed aneuploid DNA patterns. Nine tumours were classified differently by the two methods: flow cytometry resulted in diploidy in 2 and aneuploidy in 7 patients, whereas microspectrophotometry showed diploidy in 5 tumours, tetraploidy in 3 and aneuploidy in 1. Methodological limitations may explain the discrepancy in the results obtained by the two methods. However, both the flow cytometry and the microspectrophotometry method show the presence of aneuploid DNA patterns in GH-producing pituitary adenomas despite their benign growth characteristics and the clinically benign course of the disease. This comparative study with two methods measuring DNA content, shows that depending on the criteria used for diploidy-aneuploidy, the frequency of aneuploidy will vary. In this material of 26 GH-producing adenomas, 46% were aneuploid according to flow cytometry and 23% according to microspectrophotometric. However, no correlation to tumour size or GH levels was found with either method when patients with aneuploid and diploid tumours were compared. Therefore, no clinical significance can so far be drawn from these results.

Acromegaly↗
PubMed2000

Traumatic brain injury alters the molecular fingerprint of TUNEL-positive cortical neurons In vivo: A single-cell analysis.

The cerebral cortex is selectively vulnerable to cell death after traumatic brain injury (TBI). We hypothesized that the ratio of mRNAs encoding proteins important for cell survival and/or cell death is altered in individual damaged neurons after injury that may contribute to the cell's fate. To investigate this possibility, we used amplified antisense mRNA (aRNA) amplification to examine the relative abundance of 31 selected candidate mRNAs in individual cortical neurons with fragmented DNA at 12 or 24 hr after lateral fluid percussion brain injury in anesthetized rats. Only pyramidal neurons characterized by nuclear terminal deoxynucleotidyl transferase-mediated biotinylated dUTP nick end labeling (TUNEL) reactivity with little cytoplasmic staining were analyzed. For controls, non-TUNEL-positive neurons from the cortex of sham-injured animals were obtained and subjected to aRNA amplification. At 12 hr after injury, injured neurons exhibited a decrease in the relative abundance of specific mRNAs including those encoding for endogenous neuroprotective proteins. By 24 hr after injury, many of the mRNAs altered at 12 hr after injury had returned to baseline (sham-injured) levels except for increases in caspase-2 and bax mRNAs. These data suggest that TBI induces a temporal and selective alteration in the gene expression profiles or "molecular fingerprints" of TUNEL-positive neurons in the cerebral cortex. These patterns of gene expression may provide information about the molecular basis of cell death in this region after TBI and may suggest multiple avenues for therapeutic intervention.

Animals↗
PubMed2006

Single cell analysis of phosphoinositide 3-kinase/Akt and ERK activation in acute myeloid leukemia by flow cytometry.

BACKGROUND AND OBJECTIVES: Abnormal activation of several signal transduction pathways such as phosphoinositide 3-kinase (PI3K) and MAP kinases has been reported in acute myeloid leukemia (AML). To test new targeted therapeutics, it is critical to develop sensitive analytical tools to detect abnormal activation of these pathways and to monitor their inhibition in response to treatment. DESIGN AND METHODS: We analyzed Akt and ERK phosphorylation in 32 samples from patients using western blot and a two-color flow cytometry protocol using CD34. To circumvent the CD34 negative AML found in our series, we developed a two-color protocol using CD45 to isolate the blast cell population. Finally, a four-color protocol was used to detect phosphorylation in an enriched population of AML stem cells. RESULTS: We compared western blot analysis and flow cytometry for the detection of PI3K/Akt and ERK activation and found a 100% correlation between the two techniques in a series of 32 AML samples. Using a flow cytometry protocol, we were able to analyze all the patients' samples, even those with low blast infiltration or CD34 negative blast cells. We were also able to detect the phosphorylated proteins in the most immature blast cell population with the CD34+ CD38-/low CD123+ phenotype. Interpretations and CONCLUSION: Our study shows that flow cytometry is a reliable method for detecting Akt and ERK phosphorylation in all patients' samples. Activation can also be detected in the most immature blast cells, which represent exquisite target cells for new therapeutics.

Acute Disease↗
PubMed1986

Single cell analysis of calcium mobilization in anti-immunoglobulin-stimulated B lymphocytes.

A rapid increase in the concentration of intracellular free calcium ([Ca++i]) in B cells after mIg crosslinking has been documented previously by fluorimetric analysis of cell populations loaded with the fluorescent Ca++ indicator Quin 2. Although providing a valuable indication of Ca++ mobilization in the population as a whole, it has not been possible to determine whether only a subpopulation of the cells or the entire population exhibits this response. In this report, we describe the marriage of flow cytometry and Quin 2 technology, which permits discrimination of Ca++ mobilization by subpopulations of cells, as well as in whole populations. We have determined that the entire mIg+ population exhibits a synchronous increase in [Ca++i] rapidly after stimulation. Furthermore, all members of the population appear to undergo an approximately equal response.

Aminoquinolines↗
PubMed1989

Single cell analysis of DNA modifications induced by chemical carcinogens and cytostatic drugs.

Antibodies recognizing specific DNA modifications allow the immunocytochemical visualization and quantification of these modifications at the level of the individual cell. Thus, the formation and repair of DNA adducts induced by chemical mutagens and carcinogens and by cytostatic drugs can be studied in very small samples in relation to e.g. cell type and tissue localization. A number of existing and potential applications in the fields of chemical carcinogenesis, chemical mutagenesis, experimental chemotherapy and molecular epidemiology are illustrated.

Animals↗
PubMed1989

A single-cell analysis of early retinal ganglion cell differentiation in Xenopus: from soma to axon tip.

Intracellular injections of Lucifer yellow (LY) were made into the cell bodies of Xenopus retinal ganglion cells from the earliest stages of axonogenesis to the beginning of target innervation. Embryos were intact during the injection so that the entire cell (cell body, dendrites, axon, and growth cone) could be visualized. The purpose of the study was 3-fold: (1) to characterize the early steps in retinal ganglion cell differentiation before the axon reaches its target; (2) to determine whether guidepost cells exist as possible navigation cues in the vertebrate optic pathway; and (3) to investigate whether the morphology of early retinal ganglion cell growth cones varies in a position-dependent manner along the primordial optic pathway. Axons were generally initiated before dendrites and followed a well-defined course along the primordial optic pathway without branching. Surprisingly, at least 5% of the retinal ganglion cells sent more than one axon into the optic pathway. Sister axons from the same parent cell traveled separately in the pathway, indicating that their growth cones navigated independently. Examination of dendrite genesis showed that dendrites usually begin to emerge from the cell body well before the axon tip reaches the target. This observation argues against the possibility that target contact influences dendrite initiation. Nascent dendrites were commonly tipped with pronounced varicosities that did not resemble axon growth cones. Their number and branching correlated well with axon length, indicating that the age of the retinal ganglion cell itself, rather than the age of its presynaptic cells or local environment, is the strongest influence on dendrite genesis. Examination of LY-filled growth cones at varying points in the pathway showed no evidence of dye transfer to adjacent cells. This indicates that gap junctional contacts probably do not form during axonal pathfinding and suggests that direct intercellular communication between growing axons and other cells in the pathway does not play a major role in axon guidance. Growth cone morphology was analyzed quantitatively and found to vary at different positions along the pathway. Growth cones entering the optic nerve head were the largest and most complex; those on the retinal surface were the smallest and showed a simple morphology. Growth cones in the chiasm and optic tract showed a degree of complexity similar to those in the optic nerve head but were smaller.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1979

Single cell analysis of changes in electrokinetic properties of a growing ascitic tumor.

An ascitic tumor (SEWA) induced by polyoma virus in A.SW mice was analyzed in vivo as well as in vitro with regard to the electrophoretic mobility (EPM) which may be considered as a reliable criterion of surface charge. After the i.p. transplantation of 10(5) cells, the EPM decreased up to 14th day. Then, the mobility gradually increased with the age of the tumor. In the first phase of tumor growth, we have considered the possibility that immunoglobulin cell coating may be responsible for the decrease in EPM. In the late phase of SEWA growth, the progressive increase in EPM might be due to a rearrangement of sialic acids on the outer part of the cell membrane.

Animals↗