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PubMed1995

A critical assessment of the use of microculture tetrazolium assays to measure cell growth and function.

Microculture tetrazolium assays (MTAs) are being widely applied to probe the relationships between cell survival, growth, and differentiation and also to investigate associations between compromised cell metabolism, oxidative stress, and programmed cell death as occurs in apoptosis. MTAs rely upon the cellular reduction of tetrazolium salts to their intensely coloured formazans. The resulting colorimetric assays form the basis of exceptionally precise systems which are technically amenable and capable of a high throughput of samples. As a consequence, MTAs are being used to monitor responses to both extracellular activators and toxic agents in disciplines as diverse as radiobiology and endocrinology. We review the chemistry and histochemical applications of tetrazolium salts and subsequently discuss the criteria for their use in MTAs. These assays are one of the latest examples of the application of the tetrazolium/formazan system to cell biology. We outline current views on the mechanisms of the bioreduction of tetrazolium salts. These probably combine to reflect the integrated pyridine nucleotide dependent redox state of the cell. We try to illustrate how an understanding of these mechanisms helps to avoid some of the pitfalls of the MTA systems. There is now for example, extensive evidence that changes in cell culture environments, such as glucose supply or pH of the medium, influence the reduction of tetrazolium salts and thereby introduce artefacts into MTAs. Finally, we provide examples of situations in which MTAs can be used to complement other more established experimental systems. They then act as unique probes with which to investigate changes in the redox state of the cell. These changes are associated with regulation of cell growth, proliferation and differentiation and conversely, the different pathways leading to cell death.

Animals↗
PubMedDate not supplied

Rapid determination of neomycin by a microbiological agar diffusion assay using triphenyltetrazolium chloride.

The standard, quantitative determination of neomycin activity by the agar diffusion method requires an 18 to 24 h incubation time. To reduce incubation time, an alternative method using triphenyltetrazolium chloride has been developed. The inhibition zone appears much earlier; hence, an effort was made to standardize it. This indicator develops a physical response, related to the biological one, after a determined period. A Staphylococcus epidermidis suspension inoculated into solid medium at a concentration low enough that growth is not visible can reduce the dye to formazan. There is enough contrast to read the inhibition zone optically after a 7 h incubation. There is no significant difference between standard curves for 7 and 24 h incubations. Comparative assays for some pharmaceutical drugs containing neomycin in different forms show that it is possible to reduce incubation time. This modification is valid because no dispersion of results was detected by statistical analysis, which indicates that they belong to the same population. Thus, a rapid microbiological assay of neomycin has been validated and standardized.

Agar↗
PubMed1997

Substance P stimulates vascular endothelial cellular reducing capacity in the presence of insulin and human plasma factors.

Substance P (SP) is an important tachykinin in vascular wall biology. In previous studies [Villablanca et al. (1994): Circ Res 75:1113-1120], the authors have demonstrated that SP is a stimulus for endothelial cell growth and proliferation in serum-free culture conditions with cell quiescent in the G0-G1 phase of the cell cycle. As mitogenic and metabolic activity may interrelate, the purpose of this study was to determine the effects of the vasoactive perivascular neuropeptide SP on changes in the metabolic function of endothelial cells, and to characterize the response, by studying cellular reducing capacity in aortic vascular endothelial cells. In addition, interactions between SP and other growth factors (insulin and non-platelet plasma factors) were investigated and compared to the responses to SP alone. Metabolic effects were determined by evaluating cellular reducing capacity by the conversion of (3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide) to formazan (the MTT assay). The findings demonstrated that SP alone (10 pg/ml-25 micrograms/ml) inhibited cellular reducing capacity in vascular endothelial cells. In contrast, SP in the presence of insulin (10 micrograms/ml) stimulated endothelial reducing capacity, as compared to SP alone, by twofold on average. The effect of SP and insulin was additive at < or = 0.001 microgram/ml SP, and synergistic at SP concentrations ranging within 0.01-1.0 microgram/ml. SP in the presence of human platelet-poor plasma (HPPP, 5%) stimulated endothelial reducing capacity, as compared to SP alone, by threefold on average. The effect of SP and HPPP was additive at < or = 0.01 microgram/ml SP and synergistic at SP concentrations of 0.1-25 micrograms/ml. Lastly, SP in the presence of insulin and HPPP stimulated endothelial metabolic activity, as compared to SP alone, by 14-fold on average. An additive response to SP, insulin, and HPPP was observed at the lowest SP concentration studied (10 pg/ml). At all other SP concentrations studied (0.0001-25 micrograms/ml), the responses to insulin, HPPP, and SP were synergistic. Our studies indicate that the vasoactive neuropeptide substance P may synergize with insulin and HPPP in regulating endothelial cell metabolism. In addition, our findings suggest that the mechanisms by which SP stimulates cellular metabolism are different from the mechanisms by which it stimulates cell growth.

Animals↗
PubMed1998

Cytotoxicity of urethane dimethacrylate composites before and after aging and leaching.

The in vitro cytotoxicity of urethane dimethacrylate composites cured at different times by visible light and after different aging times and extraction treatments was evaluated using succinic dehydrogenase activity in the mitochondria of a fibroblastic cell line to reflect cell viability. In addition, extractable chemicals associated with cell response were identified. The composite samples were tested untreated, polished, or extracted with water or 75% ethanol-water. Balb/c 3T3 fibroblasts were used as the cell culture system while MTT-formazan production was used as the toxicity parameter. Cell viability was calculated as a percentage of Teflon controls. Identification of the chemicals was measured by extracting the composites with 75% ethanol-water, separating the extract by HPLC, and identifying the fractions with mass spectroscopy. In general, cell viability increased continuously with curing time for differently treated samples at high aging times (288 h) while it decreased when the composites were not aged (0 h). In addition, for 75% ethanol or water-extracted composites, cell viability increased within the first 24 h of aging and reached a plateau after 72 h. Lowest cytotoxicity occurred when the samples were extracted with the 75% ethanol solution. The highest cytotoxic effects were found when the samples were untreated. Slightly reduced cytotoxic effects were seen with polished composites. The results suggest that curing the light-activated composites for a minimum of 150 s and post-curing for 24 h is required to attain comparable biocompatibility with the Teflon control. Removing the oxygen-inhibited layer from these composites decreased the cytotoxicity by 33% while extracting the composites with 75% ethanol-water decreased it by 77%. Chemicals released from the surface accounted for approximately 40% of cellular response while about 60% of the response was due to chemical components released from the bulk. The primary leachable component from the composites was UDMA monomer. Small quantities of 1,6 hexane diol methacrylate, camphoroquinone, and 2,4,6-tritertiarybutyl phenol also were found.

3T3 Cells↗
PubMed1999

Visualization of the enzyme trimethylamine oxide demethylase in isoelectric focusing gels by an enzyme-specific staining method.

An enzyme-specific staining method for trimethylamine oxide demethylase (TMAO-ase) was developed. Direct visualization could be reached by coupling the reactions of the specific TMAO-ase assay with another reaction step generating as final product a dark-blue formazan. For these purposes 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) as tetrazolium salt and phenazine methosulfate (PMS) as electron transfer substance were used. Clear, dark-blue colored bands could be detected on 300 microm isoelectric focusing gels (IEF). Comparisons of enzyme-stained and protein-stained gels showed that diffusion could not be observed and that the band pattern of TMAO-ase could also be seen in the protein stain. The pI range where TMAO-ase was located was 5.6-6.6 for extracts and 6.2-6.6 for partially purified TMAO-ase. Specificity of stained TMAO-ase bands was assessed by the preparation of staining solution without the substrate trimethylamine oxide (TMAO) and by extraction of TMAO-ase from the gel and performance of the specific TMAO-ase assay.

Aldehyde-Lyases↗
PubMed1990

An in vitro chemosensitivity test for the screening of anti-cancer drugs in childhood leukemia.

We applied the MTT dye reduction assay to the anti-cancer drug sensitivity test using short-term microplate cultures. Blast cells were cultured with approximately 25 anti-cancer drugs for 4 days. After cultivation, MTT dye was placed in each well, and the formazans generated by living cells were dissolved in acidified isopropyl alcohol. The absorbance of each well was measured at a scanning microplate photometer. When we made the table of the cytotoxicity index (CI) that was classified into anti-cancer drugs and concentrations for each leukemic sample, it was possible to compare efficacy with different drugs and to select the effective ones. Retrospectively, the in vitro results were compared with the clinical responses of the 34 patients (26 of acute lymphocytic leukemia [ALL] and eight of acute nonlymphoblastic leukemia [ANLL]) who were treated by combination chemotherapy. The following results were obtained: true-positive rate, 78.1%; true-negative rate, 57.1%; and predictive accuracy, 74.4%. Therefore, the MTT assay-CI table might serve as a reliable tool for the selection of effective chemotherapy in patients with acute leukemia.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed2000

Sulfur mustard induces apoptosis and necrosis in SCL II cells in vitro.

Sulfur mustard (bis(2-chloroethyl) sulfide, HD) is an alkylating agent causing erythema and blistering with a latency of several hours after skin exposure. In the present in vitro study the influence of HD (1 microM-1 mM for 30 min or 4 h) on the viability and growth of SCL II cells was investigated. No significant differences in cytotoxicity were observed as assessed by formazan formation from XTT tetrazolium salt at 24, 48 and 72 h after exposure. Sulfur mustard concentrations of >500 microM were associated with an increasing portion of apoptotic cells without change in necrosis rate as assessed by nuclear morphology and gel electrophoresis of the DNA. The ATP levels were not affected up to 6 h after HD exposure (< or =1 mM). Twelve hours later, ATP depletion was observed at HD concentrations of >500 microM. Colony-forming ability was impaired at concentrations of <1 microM. Cell growth studies in comparison with nuclear morphology indicated late apoptotic death predominating at lower concentrations of HD. In summary, the data show that HD may inhibit cell growth already at concentrations where viability parameters and cell metabolism are not yet affected.

Adenosine Triphosphate↗
PubMed2004

Caseinophosphopeptides and their cell modulating potential.

In the context of the EU research project FAIR-CT 98-3077, studies were carried out to investigate caseinophosphopeptides (CPP) as potential ingredients for functional food and pharmaceutical applications. CPP preparations were produced by enrichment of CPP from hydrolytic casein digests. Enzyme preparations used for hydrolysis were PTN 3.0 S, Alcalase, Bioprotease P conc, Proteinase DS. Cytochemical studies were carried out to examine the cytotoxic potential or cell modulating activities of CPP using human cancer cell lines (HL-60, Caco-2) and non-malignant polymorphonuclear leukocytes (PML) from oral cavity. PML cells isolated by magnetic cell sorting using CD-15-antibody-labelled paramagnetic beads were used for the first time for testing food-derived peptides. Cell activity was measured by formazan dye formation. Effects on enterocytic differentiation properties of Caco-2 cells were examined by transepithelial membrane resistance of Caco-2 cell monolayers and brush border associated alkaline phosphatase activity. In conclusion, (1) no deleterious cytochemical consequences (apoptotic, antiproliferative or general cytotoxic effects) were observed on presenting a range of CPP preparations to various human cell systems, indicating that these compounds can be rated harmless at a cellular level, (2) stimulation of IgG-secretion into culture supernatant of PBL points to possible immunoenhancing properties of CPP preparations.

B-Lymphocytes↗
PubMed2006

Effect of spatial architecture on cellular colonization.

The spatial cell-material interaction remains vital issue in forming biodegradable scaffolds in Tissue Engineering. In this study, to understand the influence of spatial architecture on cellular behavior, 2D and 3D chitosan scaffolds of 50-190 kD and >310 kD MW were synthesized through air drying and controlled rate freezing/lypohilization technique, respectively. In addition, chitosan was emulsified with 19, 76, and 160 kD 50:50 poly lactide-co-glycolide (PLGA) using 1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine (DMPC) as stabilizer. 2D and 3D scaffolds were formed by air drying and lyophilization as before. Tensile and compressive properties of films and scaffolds were analyzed in wet conditions at 37 degrees C. Alterations in the cell spreading, proliferation, and cytoskeletal organization of human umbilical vein endothelial cells (HUVECs) and mouse embryonic fibroblasts (MEFs) were studied. These results showed that the formed 3D chitosan scaffolds had interconnected open pore architecture (50-200 microm size). HUVECs and MEFs had reduced spreading areas and circular morphology on 2D chitosan membranes compared with 3D chitosan scaffolds. The fluorescence photomicrographs for actin (using Alexa Fluor 488 phalloidin) and cytoplasm staining (using carboxyfluorescein diacetate-succinimidyl ester) demonstrated that the cells spread within 3D chitosan matrix. 2D and 3D emulsified chitosan and chitosan/PLGA scaffolds reduced the spreading of HUVECs and MEFs even further. Proliferation results, analyzed via MTT-Formazan assay and BrdU uptake assay, correlated with the spreading characteristics. The reductions in cell spreading area on emulsified surfaces were not detrimental to the viability and endocytic activity but to proliferation. The observed alterations in cellular colonization are in part due to the substrate stiffness and surface topography. In summary, these results suggest a significant influence of spatial architecture on cellular colonization.

Cell Culture Techniques↗
PubMed2005

Detection and purification of glucose 6-phosphate dehydrogenase, malic enzyme, and NADP-dependent isocitrate dehydrogenase by blue native polyacrylamide gel electrophoresis.

We describe a blue native polyacrylamide gel electrophoretic technique that allows the facile detection, quantitation and purification of three NADPH-producing enzymes. Glucose 6-phosphate dehydrogenase, malic enzyme and NADP-dependent isocitrate dehydrogenase were detected simultaneously. Activity staining based on the formation of NADPH from the respective substrates and the subsequent precipitation of formazan enabled the relative quantitation of enzymatic activities, while Coomassie staining on one-dimensional or two-dimensional gels helped monitor the amount of protein associated with these enzymatic activities. This technique provides a simple and effective route to obtain homogeneous protein for further analyses and also enables the screening of these NADPH-producing enzymes in various cellular systems.

Electrophoresis, Polyacrylamide Gel↗
PubMed2003

Peroxynitrite-induced oligodendrocyte toxicity is not dependent on poly(ADP-ribose) polymerase activation.

Oligodendrocyte loss is a characteristic feature of several CNS disorders, including multiple sclerosis (MS) and spinal cord injury. However, the mechanisms responsible for oligodendrocyte destruction remain undefined. As recent studies have implicated peroxynitrite in the pathogenesis of both spinal cord injury and MS, we have examined whether peroxynitrite may mediate at least some of the oligodendrocyte damage and demyelination observed in these conditions. Primary rat oligodendrocytes were exposed to authentic peroxynitrite in vitro and assessed for cytotoxicity. Mitochondrial function, measured by the reduction of MTT to formazan, and mitochondrial membrane potential were used as indicators of cell viability. Cell death was quantitated by measuring either the release of lactate dehydrogenase from, or the uptake of propidium iodide into, damaged and dying cells. Peroxynitrite dose-dependently reduced the viability of primary oligodendrocytes and induced cell death. Furthermore, peroxynitrite significantly increased DNA strand breakage and the activity of poly(ADP-ribose) polymerase (PARP) in oligodendrocyte cultures. To identify whether PARP activation plays a role in peroxynitrite-induced oligodendrocyte toxicity, we examined the effects of the PARP inhibitors 3-aminobenzamide (3AB) and 5-iodo-6-amino-1,2-benzopyrone (INH(2)BP) on mitochondrial function and cell death in oligodendrocytes. The presence of 3AB and INH(2)BP did not protect oligodendrocytes from peroxynitrite-induced cytotoxicity. However, both compounds significantly reduced PARP activity in these cells. Primary oligodendrocytes generated from PARP-deficient mice were also highly susceptible to peroxynitrite-induced cell death. Therefore, our results show that peroxynitrite exerts cytotoxic effects on oligodendrocytes in vitro independently of PARP activation.

Animals↗
PubMed1994

Establishment and in vivo characterization of multidrug-resistant dunning R3327 rat prostate-carcinoma cell-lines.

We describe the selection of 3 new multidrug-resistant cell lines derived from tumor cells of different metastatic phenotypes within the Dunning R3327 model of rat prostatic carcinoma. Cell lines of weak (AT2) and strong (AT3 and MAT-LyLu) metastatic behavior were cultured in vitro and challenged with doxorubicin at progressively increasing concentrations. Chemosensitivity was determined colorimetrically by release of precipitated formazan pigment (MTT assay). Expression of the multidrug-resistance glycoprotein (P-170) was monitored immunocytochemically and by Western blotting using monoclonal antibody C219. The behavior of the parental and resultant drug-resistant cells was assessed by their growth in syngeneic rats. Doxorubicin challenge of the initially drug-sensitive parental prostatic carcinoma cell lines resulted in the rapid development of multidrug resistance together with simultaneous expression of P-glycoprotein. While lung and lymph-node metastases developed in host animals inoculated with parental AT3 and MAT-LyLu cells, no metastases developed in the multidrug-resistant progeny of these cell lines. This study has shown that Dunning rat prostate-carcinoma cell lines, previously sensitive to different cytotoxic agents, rapidly become multidrug-resistant and express P-glycoprotein following exposure to doxorubicin. Furthermore, development of multidrug resistance is associated with a less aggressive tumor phenotype and loss of metastatic potential. Nevertheless, it is unlikely that the non-metastatic phenotype of Dunning rat prostatic carcinoma cells is solely associated with expression of P-glycoprotein. These new multidrug-resistant cell lines exhibiting an altered behavioral phenotype will provide a valuable model with which to analyze the relationship between expression of P-glycoprotein and the metastatic phenotype of prostatic carcinoma cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1991

The in vitro effects of metal cations on eukaryotic cell metabolism.

The in vitro cytotoxicity of nine metal cations common in dental casting alloys was evaluated using Balb/c 3T3 fibroblasts and four toxicity parameters: total protein production, 3H-leucine incorporation, 3H-thymidine incorporation, and MTT-formazan production. Concentrations causing 50% toxicity compared to controls (TC50's) and reversibility of these effects were determined. The range of potency of the metal cations was 2-3 orders of magnitude, with Cd2+ showing the greatest potency and In3+ showing the least. Potency did not correlate with atomic weight for these metals. For each metal cation, the TC50's of the various toxicity parameters were similar in most cases. However, several cations (Cu2+, Ga3+) showed greater potency with 3H-thymidine incorporation. Reversibility of the toxic effects was observed for all cations; the effects generally became irreversible at concentrations in the range of the TC50 value for each cation. Several stimulatory effects were seen. Small but statistically significant stimulations were observed after 24 h of metal exposure for Ag1+, Au4+, Cu2+, Ga3+, and Ni2+. Residual stimulations 24 h after removal of the metal cations were observed for Au4+, Cd2+, Ni2+, and Zn2+. Stimulations always occurred at concentrations below the TC50 concentrations. This study should be useful in evaluating the potential cytotoxic effects of metal cations released from dental alloys.

3T3 Cells↗
PubMed1992

In vitro synergistic, antagonistic, and duration of exposure effects of metal cations on eukaryotic cells.

Effects of duration of exposure of single metal cations (Ag+1, Cd+2, Cu+2, Ga+3, Ni+2, and Zn+2) on 3H-thymidine (3H-Tdr) incorporation and 3-[4,5-dimethyl-thiazol-2yl-]-2,5-diphenyl tetrazolium bromide-formazan (MTT-f) production in cultured murine fibroblasts (Balb/c 3T3) were investigated, and the synergistic and antagonistic effects of two metal cations applied simultaneously to the fibroblasts were assessed. The effects of duration of exposure were quantified using TC50 values (concentration of an element required to cause 50% toxicity compared with controls) measured after 24, 48, or 72 h. Using MTT-f production, Cd+2, Cu+2, Ga+3, and Ni+2 showed significantly lower TC50 values with increasing time of exposure, whereas the TC50 values for Ag+1 and Zn+2 remained constant. The TC50 values using 3H-Tdr incorporation exhibited a similar pattern with time of exposure, but the effects were less pronounced. The TC50 values for 3H-Tdr and MTT-f tended to equalize at 72 h. All combinations of cations tested (Ag-Cu, Ag-Zn, Ag-Ni, Cu-Zn, Cu-Ni, Ga-Ni, and Cu-Cd) exhibited synergistic and antagonistic effects as measured by MTT-f production. Synergistic and antagonistic effects were not necessarily mutually exclusive in the same system.

3T3 Cells↗
PubMed1995

Assessment of viability and proliferation of in vivo silicone-primed lymphocytes after in vitro re-exposure to silicone.

The functional response of peripheral blood lymphocytes isolated from 22 patients with silicone gel-filled breast implants was assessed after in vitro re-exposure to silicone. Using cell culture test methods to quantify proliferation and viability and/or activation of lymphocyte microcultures, i.e., the uptake of tritiated thymidine (3H-TdR uptake test) and the reduction of formazan salts (MTT assay), interesting data were obtained. Peripheral blood lymphocytes purified from patients wearing silicone gel-filled breast implants react in vitro to silicone showing a statistically significant increase of both proliferation and viability, while healthy subjects do not respond on in vitro exposure to silicone. Differences resulted even more statistically significant when patients were divided into two groups depending on the type of surgery they underwent: patients with breast augmentation for aesthetic reasons seem to have an increased responsiveness in vitro to silicone compared to patients who experienced a reconstructive surgery of the breast. Although they are still preliminary, being referred to a limited population, these results suggest that the lymphocytes of patients with silicone gel-filled breast implants could be sensitized in vivo toward silicone; the re-exposure of these cells to silicone leads to a higher functional response which could be looked for by using quantitative in vitro test methods.

Adult↗
PubMed1992

Nitroblue tetrazolium reduction and bone resorption by osteoclasts in vitro inhibited by a manganese-based superoxide dismutase mimic.

Oxygen-derived free radicals are produced by osteoclasts. Oxygen radical formation occurs at the osteoclast/bone surface interface. This location next to bone implies that oxygen radicals, including but not limited to superoxide, are needed for bone resorption. Compounds that scavenge superoxide are being developed as pharmaceutical agents to inhibit the damaging effects of oxygen radical formation on tissues. One such scavenger is the Desferal-manganese complex (DMnC). DMnC reduced the amount of formazan staining produced by the interaction of oxygen radicals with nitroblue tetrazolium (NBT) in both individual mouse calvarial osteoclasts in tissue explants and isolated osteoclasts. As a result of the reduced concentrations of oxygen radicals, DMnC inhibited bone resorption by calvarial explants and isolated osteoclasts. Superoxide dismutase (SOD) inhibited NBT reduction and bone resorption by isolated osteoclasts but to a lesser degree than DMnC. Inhibition of bone resorption in the isolated osteoclast system increased in parallel to the concentration of DMnC in cultures. Desferal without Mn had no effect on bone resorption by isolated osteoclasts. These results support the hypothesis that osteoclasts produce oxygen radicals as part of the process of bone resorption.

Analysis of Variance↗
PubMed2005

Nicotinic and PDGF-receptor function are essential for nicotine-stimulated mitogenesis in human vascular smooth muscle cells.

Cigarette smoking is implicated in the formation of occlusive vascular diseases. Nicotine's role in this process is incompletely understood. Nicotine's effect on human aortic vascular smooth muscle cells (HaVSMC) and the role of the nicotinic receptor (nAChR), platelet-derived growth factor (PDGF), and the PDGF-receptor (PDGF-R) in this response were studied. Nicotine's mitogenic effect was characterized by three methods: thymidine incorporation, a viability/proliferation assay based on metabolic conversion of tetrazolium salt to formazan dye and cell counting. Nicotine administration (10(-6) M) stimulated cell cycle entry marked by increased DNA synthesis, PCNA and cyclin D1 production, and increased cell division. Nicotinic receptor blockade with d-tubocurarine, a nicotinic AchR blocker, decreased nicotine-induced DNA synthesis, and cell division (0.33 +/- 0.04, 0.77 +/- 0.31-fold decrease, respectively). Nicotine increased cellular PDGF-BB transcript levels and protein release (ELISA: 1.6 +/- 0.5-fold increase) but not PDGF-AA or PDGF-AB release. Nicotine increased PDGFbeta-receptor protein content. PDGF inactivation with anti-PDGF antibody abolished nicotine-induced DNA synthesis (1.9 +/- 0.08-fold decrease). PDGF-R blockade with the PDGF-R antagonist tyrphostin AG 1295 decreased nicotine-induced DNA synthesis and cell division (0.25 +/- 0.01, 0.44 +/- 0.2-fold decrease, respectively). PDGF-R blockade reversed nicotine-stimulated increases in PDGF release, PDGF-BB transcripts, and PDGF-receptor levels (0.68 +/- 0.34; 0.46 +/- 0.01; 0.28 +/- 0.01-fold decrease, respectively). In conclusion, nicotine-mediated activation of nAChRs increases PDGF-BB transcription and protein production as well as PDGF beta-receptor levels. PDGF-BB/PDGF-R interaction is vital in nicotine's mitogenic actions on human aortic smooth muscle cells.

Adult↗
PubMed2002

Evaluation of in vitro proliferative activity of human fetal neural stem/progenitor cells using indirect measurements of viable cells based on cellular metabolic activity.

To scale up human neural stem/progenitor cell (NSPC) cultures for clinical use, we need to know how long these cells can live ex vivo without losing their ability to proliferate and differentiate; thus, a convenient method is needed to estimate the proliferative activity of human NSPCs grown in neurosphere cultures, as direct cell counting is laborious and potentially inaccurate. Here, we isolated NSPCs from human fetal forebrain and prepared neurosphere cultures. We determined the number of viable cells and estimated their proliferative activity in long-term culture using two methods that measure viable cell numbers indirectly, based on their metabolic activity: the WST-8 assay, in which a formazan dye is produced upon reduction of the water-soluble tetrazolium salt WST-8 by dehydrogenase activity, and the ATP assay, which measures the ATP content of the total cell plasma. We compared the results of these assays with the proliferative activity estimated by DNA synthesis using the 5-bromo-2'-deoxyuridine incorporation assay. We found the numbers of viable human NSPCs to be directly proportional to the metabolic reaction products obtained in the WST-8 and ATP assays. Both methods yielded identical cell growth curves, showing an exponentially proliferative phase and a change in the population doubling time in long-term culture. They also showed that human NSPCs could be expanded for up to 200 days ex vivo without losing their ability to proliferate and differentiate. Our findings indicated that indirect measurements of viable cells based on metabolic activity, especially the ATP assay, are very effective and reproducible ways to determine the numbers of viable human NSPCs in intact neurospheres.

Adenosine Triphosphate↗