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PubMedDate not supplied

Comparison of trypan blue dye exclusion and fluorometric assays for mammalian cell viability determinations.

A hemocytometer-based trypan blue dye exclusion cell quantitation and viability assay was compared with a similar assay using simultaneous fluorometric staining with fluorescein diacetate and propidium iodide. Viable and nonviable cell densities were measured, and culture viability was calculated both during the normal growth cycle of a murine hybridoma and in response to the application of millimolar concentrations of either tert-butyl hydroperoxide or ferrous iron. During the early phase of rapid hybridoma cell growth, assay-based differences in viable cell density were not significant. As the culture aged, the trypan blue dye exclusion assay significantly overestimated cell viability, thereby underestimating nonviable cell density and yielding an erroneous estimation of the overall viability of the culture. Because of its lack of ambiguity in the identification of stained, nonviable cells and its resulting increased accuracy in the estimation of culture viability, the fluorometric assay was considered a better choice for the evaluation of cell viability.

Animals↗
PubMed1992

Ciliary function, cell viability, and in vitro effect of ribavirin on nasal epithelial cells in acute rhinorrhea.

Nasal epithelial (NE) cells were collected from the nasopharynx of 25 individuals with symptomatic colds and 27 healthy volunteers (controls), and ciliary beat frequency (CBF) was assessed by microscopy employing video motion analysis techniques. Baseline CBF was statistically significantly elevated in the group with colds compared to the control group (14.6 +/- 1.5 Hz [mean +/- SD] vs 13.8 +/- 0.9 Hz; p = 0.02). After four days of incubation in culture, there was a significant decrease in the CBF in both groups, with a change from baseline of 1.9 Hz for the cold group, compared to 1.0 Hz for the control group (p = 0.0001). The in vitro addition of ribavirin at 500 micrograms/ml to NE cells from individuals with colds preserved the viability of the cells and maintained the CBF at baseline values. Twenty-four (96 percent) of 25 ribavirin-treated specimens from the cold group survived for four days in culture, compared with 17 (68 percent) of 25 untreated cold specimens. In addition, the ribavirin-treated cells had a mean CBF of 14.2 +/- 1.3 Hz, compared with 12.7 +/- 1.9 Hz for the untreated cell samples (p = 0.0005). Ribavirin had no effect on NE cells from the control group. These results suggest that ribavirin in a concentration of 500 micrograms/ml may have some benefit in the treatment of acute rhinorrhea.

Acute Disease↗
PubMed1991

Interleukin-4 enhances anti-IgM stimulation of B cells by improving cell viability and by increasing the sensitivity of B cells to the anti-IgM signal.

The lymphokine IL-4 is a potent enhancer of anti-IgM-induced B cell proliferation. Although the mechanism of this enhancement is not known, a commonly held view suggests that IL-4 acts together with anti-IgM as a costimulating factor for the activation of a subpopulation of B cells. To evaluate this hypothesis we examined the effect of IL-4 on the proportion of B cells stimulated to divide by different doses of anti-IgM using flow cytometry in combination with measurements of tritiated-thymidine incorporation. The results suggest a novel and surprisingly simple model for the mode of action of IL-4. Our analysis revealed that at high saturating anti-IgM concentrations, the proportion of live B cells which enter into S phase of the cell cycle is the same (approximately 65%) for cells cultured with or without IL-4. Cultures containing IL-4, however, exhibit a twofold increase in thymidine uptake over cultures without IL-4. This increase can be explained completely by the ability of IL-4 to enhance the viability of small dense B cells over the first 24 hr from approximately 50 to 90% of the starting cell number. Normalizing the maximum response levels obtained with and without IL-4 reveals that B cells incubated with IL-4 exhibit a 10-fold decrease in the concentration of anti-IgM required to stimulate the half-maximum proliferation level. Furthermore, evaluation of the number of cells in S phase by flow cytometry and analysis of the kinetics of cell proliferation revealed that the increased response effected by IL-4 at lower anti-IgM concentrations was due to a greater number of proliferating B cells rather than the same number of cells undergoing a faster division rate. We also found a highly nonlinear relationship between B cell number and proliferative response, implying a requirement for an additional, cell cooperation-mediated, activating signal for maximum B cell proliferation. IL-4 enhanced proliferation by the same proportion at all cell concentrations indicating that it does not replace or alter this requirement for cell cooperation. Taken together these results suggest that anti-IgM in combination with a second unidentified cell-cooperation-dependent signal leads to proliferation of up to 65% of small resting B cells. IL-4 does not provide an essential activation signal but serves to raise the sensitivity of B cells to the anti-IgM-generated signal.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1987

Intracellular adenosine triphosphate as a measure of human tumor cell viability and drug modulated growth.

Adenosine triphosphate is the primary energy unit for cells, and levels of this compound offer a potential marker for cell viability and growth. The availability of a bioluminescence assay allows for a rapid, sensitive, and reproducible measurement of ATP. A method is described for the quantification of intracellular ATP levels in human cancer cells. ATP levels were linearly related to the number of viable cells and increased with time in human cancer cell line cultures correlating with growth kinetics. The effect of 5-fluorouracil, doxorubicin, methotrexate, cytosine arabinoside, nitrogen mustard, melphalan, vinblastine, and cisplatin on the growth of human cancer cell lines was studied utilizing ATP levels. ATP levels and colony formation in agar of drug-exposed cells were compared. Overall there was a significant correlation between drug effects on colony formation and ATP levels. The ATP assay is rapid, simple, reproducible, and a relatively inexpensive method of quantifying drug effects on malignant cells. This makes it a potentially useful method for screening new anticancer drugs in human cancer cell lines.

Adenosine Triphosphate↗
PubMed1995

Endotoxin-induced changes of endothelial cell viability and permeability: protective effect of a 21-aminosteroid.

Endotoxic shock results in endothelial cell dysfunction and oedema formation. Endotoxin decreased in a concentration-dependent fashion endothelial cell viability with maximum effects by adding 10 micrograms/ml lipopolysaccharide for 48 h (47 +/- 15% and 22 +/- 2.6% of control cells in the presence or absence of foetal calf serum, respectively). Furthermore, incubation (10 h) with lower concentrations of lipopolysaccharide (1 microgram/ml) significantly increased endothelial cell permeability to 250% compared to control values. The 21-aminosteroid U-74389G (10 microM) prevented the cytotoxic effect of lipopolysaccharide as well as the lipopolysaccharide-induced increase in endothelial cell permeability. By contrast, the glucocorticoid methylprednisolone was less effective even at higher concentrations (100 microM). The effect of lipopolysaccharide is possibly due to oxidative stress and/or membrane destabilization rather than to the induction of inflammatory mediators, because of the reduced efficacy of the glucocorticoid.

Allopurinol↗
PubMedDate not supplied

Transfection of neuroprogenitor cells with iron nanoparticles for magnetic resonance imaging tracking: cell viability, differentiation, and intracellular localization.

PURPOSE: Magnetic resonance imaging (MRI) can track labeled cells in the brain. The use of hemagglutinating virus of Japan envelopes (HVJ-Es) to effectively introduce the contrast agent to neural progenitor cells (NPCs) is limited to date despite their high NPC affinity. PROCEDURES: HVJ-Es and Lipofectamine 2000 were compared as transfection vehicles of superparamagnetic iron oxide (SPIO). Labeled NPCs were examined for iron content, MRI signal change, and fundamental cell characteristics. Prussian Blue staining was used after differentiation to determine SPIO localization. RESULTS: HVJ-Es transfected up to 12.5 +/- 8.8 times more SPIO into NPCs. HVJ-Es do not affect cell viability or differentiation capability. Superparamagnetic iron oxide was disseminated in both the soma and neurites. CONCLUSIONS: These findings indicate that HVJ-Es are an effective vehicle for SPIO transfection of NPCs. The intracellular localization after differentiation raises the question as to the capability of MRI to distinguish cell migration from axonal or dendritic growth in vivo.

Animals↗
PubMed2000

hnRNP C is required for postimplantation mouse development but Is dispensable for cell viability.

The hnRNP C1 and C2 proteins are among the most abundant proteins in the nucleus, and as ubiquitous components of RNP complexes, they have been implicated in many aspects of mRNA biogenesis. In this report, we have characterized a null mutation induced in embryonic stem cells by insertion of the U3His gene trap retrovirus into the first intron of the hnRNP C1/C2 gene. cDNAs encoding murine hnRNP C1 and C2 were characterized, and the predicted protein sequences were found to be highly conserved among vertebrates. A human consensus sequence, generated from over 400 expressed sequence tags, suggests two revisions to the previously published human sequence. In addition, alternatively spliced transcripts, expressed only by the murine gene, encode four novel proteins: variants of C1 and C2 with either seven additional amino acids or one fewer amino acid in a region between the oligomerization and C-terminal acidic domains. The disrupted gene was transmitted into the germ line and is tightly linked to a recessive, embryonic lethal phenotype. Homozygous mutant embryos fail to develop beyond the egg cylinder stage and are resorbed by 10.5 days of gestation, a phenotype consistent with a fundamental role in cellular metabolism. However, hnRNP C1 and C2 are not required for cell viability. Embryonic stem cell lines established from homozygous mutant blastocysts did not express detectable levels of either protein yet were able to grow and differentiate in vitro, albeit more slowly than wild-type cells. These results indicate that the C1 and C2 hnRNPs are not required for any essential step in mRNA biogenesis; however, the proteins may influence the rate and/or fidelity of one or more steps.

Amino Acid Sequence↗
PubMed1994

Methylglyoxal and cell viability.

Methylglyoxal by depleting glutathione stores increased Trypan-blue uptake by the cells incubated in glucose, pyruvate and amino acids free medium. Only a transient fall of glutathione concentration without any effect on cell viability was caused by methylglyoxal when the medium was supplemented with above-mentioned compounds. The role of gamma-glutamyl-transpeptidase is discussed.

Amino Acids↗
PubMed1991

Growth and cell viability of aortic versus pulmonic homografts in the systemic circulation.

Growth characteristics and cell viability of aortic and pulmonic valve homografts in the systemic circulation were compared in a growing sheep model. Seven aortic and seven pulmonic cryopreserved homografts were implanted in the descending aortae of recipient female lambs. Of seven sheep per group, three were killed at 8 months, three at 12 months, and one at 15 months. Tissue cultures were obtained on homograft valve and root wall specimens. Male donor cells were identified by chromosome analysis. Preimplant to explant changes in pulmonic homograft external diameters at the valve annulus and sinotubular junction increased significantly more than in aortic roots. The postoperative dimensions of the distal anastomotic diameter and length of the graft increased significantly more in pulmonic than in aortic roots by time of explant. Leaflet calcification and valvular stenosis did not develop in either pulmonic or aortic homografts over the period of observation. Calcification in the root wall was significantly less in pulmonic than aortic homografts. Growth on tissue culture was obtained from over 70% of homograft specimens. Viable donor cells were demonstrated in none and 43% of aortic and pulmonic leaflets, respectively, and 71% and 57% of aortic and pulmonary arterial homograft walls, respectively. In conclusion, both aortic and pulmonic homograft valves provide freedom from calcification and stenosis in this model. Continuing expansion of pulmonic root diameters under systemic pressure might lead to late aneurysm formation. Although host cell repopulation of grafts may be advantageous, the presence of viable donor cells in leaflet tissue does not seem necessary to prevent calcific degeneration.

Animals↗
PubMed2004

The use of flow cytometry in the evaluation of cell viability of cryopreserved sperm of the marine shrimp (Litopenaeus vannamei).

Although the cryopreservation of penaeid prawn sperm or embryos has definite applications in the aquaculture industry, there is no protocol routinely used for this procedure. One of the main problems relies on the limitations for the determination of sperm cell viability. In this study, we evaluated the toxicity and cryoprotectant effect of four agents, at three different concentrations, in sperm suspension, spermatic mass, and complete spermatophore of the marine shrimp Litopenaeus vannamei. Cells were frozen by fast and slow cooling rates. After thawing, they were analyzed by optical microscopy and flow cytometry, which was also utilized to determine spermatic viability by DNA staining with propidium iodine. Considering viability by morphotype analysis, the best result was obtained when the spermatic mass was frozen by slow cooling rate in the presence of methanol (61.6%). There was a positive correlation between morphotype analysis and flow cytometry, although the percentage of viable cells was always lower when determined by the later. These results show that flow cytometry is a valuable tool to evaluate sperm cell viability in decapod species and it is more sensitive technique than optical microscopy.

Animals↗
PubMed2005

CPT1alpha over-expression increases long-chain fatty acid oxidation and reduces cell viability with incremental palmitic acid concentration in 293T cells.

To test the cellular response to an increased fatty acid oxidation, we generated a vector for an inducible expression of the rate-limiting enzyme carnitine palmitoyl-transferase 1alpha (CPT1alpha). Human embryonic 293T kidney cells were transiently transfected and expression of the CPT1alpha transgene in the tet-on vector was activated with doxycycline. Fatty acid oxidation was measured by determining the conversion of supplemented, synthetic cis-10-heptadecenoic acid (C17:1n-7) to C15:ln-7. CPT1alpha over-expression increased mitochondrial long-chain fatty acid oxidation about 6-fold. Addition of palmitic acid (PA) decreased viability of CPT1alpha over-expressing cells in a concentration-dependent manner. Both, PA and CPT1alpha over-expression increased cell death. Interestingly, PA reduced total cell number only in cells over-expressing CPT1alpha, suggesting an effect on cell proliferation that requires PA translocation across the mitochondrial inner membrane. This inducible expression system should be well suited to study the roles of CPT1 and fatty acid oxidation in lipotoxicity and metabolism in vivo.

Carnitine O-Palmitoyltransferase↗
PubMed2001

Quantitative study of electroporation-mediated molecular uptake and cell viability.

Electroporation's use for laboratory transfection and clinical chemotherapy is limited by an incomplete understanding of the effects of electroporation parameters on molecular uptake and cell viability. To address this need, uptake of calcein and viability of DU 145 prostate cancer cells were quantified using flow cytometry for more than 200 different combinations of experimental conditions. The experimental parameters included field strength (0.1-3.3 kV/cm), pulse length (0.05-20 ms), number of pulses (1-10), calcein concentration (10-100 microM), and cell concentration (0.6-23% by volume). These data indicate that neither electrical charge nor energy was a good predictor of electroporation's effects. Instead, both uptake and viability showed a complex dependence on field strength, pulse length, and number of pulses. The effect of cell concentration was explained quantitatively by electric field perturbations caused by neighboring cells. Uptake was shown to vary linearly with external calcein concentration. This large quantitative data set may be used to optimize electroporation protocols, test theoretical models, and guide mechanistic interpretations.

Biological Transport, Active↗
PubMed1992

Astrocytes support mast cell viability in vitro.

Mast cells are normally found adjacent to blood vessels in the nervous system, and have been implicated in the development of inflammatory central nervous system (CNS) diseases such as experimental allergic encephalomyelitis. To further study mast cell-CNS interactions, we have developed a model in which viable rat peritoneal mast cells can be maintained in culture for up to 30 days on a monolayer of rat astrocytes. In this microenvironment, mast cells maintain their phenotype, morphology, and ability to degranulate in response to appropriate stimuli.

Animals↗
PubMed1998

Effect of polyisobutylcyanoacrylate nanoparticles and lipofectin loaded with oligonucleotides on cell viability and PKC alpha neosynthesis in HepG2 cells.

The aim of the present study was to evaluate the inhibitory effect on protein kinase C alpha (PKC alpha) neosynthesis of antisense oligonucleotides delivered by two types of carriers. First, PKC alpha antisense oligonucleotides were associated with polyisobutylcyanoacrylate (PIBCA) nanoparticles pre-coated with cetyltrimethyl ammonium bromide (CTAB), a hydrophobic cation. Adsorption of oligonucleotides onto PIBCA nanoparticles was shown to be a saturating process. From these studies, it was possible to identify two types of particles: positively and negatively charged. Secondly, Lipofectin was used as another carrier system. These systems were incubated with HepG2 cells. Toxicity was evaluated by the MTT assay, and PKC alpha neosynthesis was determined by Western blots in conditions where nanoparticles and Lipofectin were not inducing cytotoxicity. It was observed that both mismatch and antisense oligonucleotides induced an inhibition of PKC alpha neosynthesis when loaded onto cationic or anionic nanoparticles as well as when complexed to cationic liposomes (Lipofectin). This non-specific effect was only observed in the phase of PKC alpha neosynthesis when the cells were first depleted in PKC alpha by phorbol 12-myristate beta-acetate (12-PMA) and in the absence of serum. These results strongly suggest that delivery systems, PIBCA nanoparticles or Lipofectin, containing a positively charged component (CTAB or cationic lipids), are able to induce a perturbation in the intracellular metabolic activity. In conclusion, it was shown that the commonly used strategy of oligonucleotides targeting with cationic non-viral vectors may display non-specific effects which can lead to artifactual results.

Adsorption↗
PubMed2003

Post-translational import of the prion protein into the endoplasmic reticulum interferes with cell viability: a critical role for the putative transmembrane domain.

Aberrant folding of the mammalian prion protein (PrP) is linked to prion diseases in humans and animals. We show that during post-translational targeting of PrP to the endoplasmic reticulum (ER) the putative transmembrane domain induces misfolding of PrP in the cytosol and interferes with its import into the ER. Unglycosylated and misfolded PrP with an uncleaved N-terminal signal sequence associates with ER membranes, and, moreover, decreases cell viability. PrP expressed in the cytosol, lacking the N-terminal ER targeting sequence, also adopts a misfolded conformation; however, this has no adverse effect on cell growth. PrP processing, productive ER import, and cellular viability can be restored either by deleting the putative transmembrane domain or by using a N-terminal signal sequence specific for co-translational ER import. Our study reveals that the putative transmembrane domain features in the formation of misfolded PrP conformers and indicates that post-translational targeting of PrP to the ER can decrease cell viability.

Amino Acid Sequence↗
PubMed1995

Cell viability of aortic allografts after long-term cryopreservation and clinical application to aortic root replacement in a patient with aortitis.

A key point in the establishment of a tissue cryopreservation system is to maintain cell viability for an extended period. The viability of the tissue cells in the authors' cryopreservation system was examined, and the first clinical application reported. The tissue culture method demonstrated 95% of rat aortas (21 of 22 specimens) and 88% of human cardiac valves (seven of eight) to be viable after 6.4 months and 5.8 months of cryopreservation, respectively. The first clinical case, a 54-year-old man with aortic valve replacement and complications of aortitis, underwent successful aortic root replacement. Whereas the use of allografts in aortitis cases is controversial, such an approach was assumed reasonable because the mechanical stress between the native aortic annulus and the inserted valve could be reduced by aortic root replacement with the allograft.

Adult↗
PubMed1997

Dual staining assessment of Schwann cell viability within whole peripheral nerves using calcein-AM and ethidium homodimer.

A membrane permeant nucleic acid stain, ethidium homodimer was used in combination with calcein-AM to document the viability of Schwann cells (SCs) in whole nerves after cold storage assays. Segments of peripheral nerves were, (i) kept intact in buffer (viability controls), (ii) thawed after a cryopreservation, according to a protocol which has been previously shown to maintain the integrity of most nerve components [Ruwe and Trumble, J. Reconstr. Microsurg., 1990, 6: 239-244; Gauthier et al., In 3rd International Symposium on Axonal Regrowth in the Mammalian Spinal Cord and Peripheral Nerve, Deauville, France, 1995, p. 24, abstract], (iii) killed by chemical injury, or (iv) by successive freezing-thawing. Teased preparations of nerve fibers were prepared from the various types of nerve segments and incubated with calcein-AM and ethidium homodimer, which stain, respectively, living and dead cells. In control or cryopreserved nerves, staining with calcein-AM resulted in bright green fluorescence in the cytoplasm of SCs, with no red fluorescence of ethidium homodimer. In contrast, in killed nerve preparations, intense ethidium red fluorescence was observed in SC nuclei, with negligible green calcein cytoplasmic fluorescence. Thus, the combination of calcein-AM/ethidium homodimer appeared as an effective tool for assessing the viability of SCs and determine the quality of cold stored nerve preparations used in graft repair procedures. In addition, the generated fluorescence enabled clear visualization of myelinated fibers by confocal imaging.

Animals↗