Use of an aqueous soluble tetrazolium/formazan assay to measure viability and proliferation of lymphokine-dependent cell lines

Buttke, Thomas M.; McCubrey, James A.; Owen, Terence C.

doi:10.1016/0022-1759(93)90092-l

Cited by 300 publications

(198 citation statements)

References 19 publications

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“…Cell Proliferation Assay and Coulter Counting-Proliferation was measured using the Cell Titer 96 AQ ueous nonradioactive cell proliferation assay (Promega, Inc., Madison, WI) (16). This assay is based on the cellular conversion of the colorimetric reagent, MTS (3,4-(5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt), into soluble formazan by dehydrogenase enzymes found only in metabolically active, proliferating cells.…”

Section: Methodsmentioning

confidence: 99%

Role of Janus Kinase/Signal Transducer and Activator of Transcription and Mitogen-activated Protein Kinase Cascades in Angiotensin II- and Platelet-derived Growth Factor-induced Vascular Smooth Muscle Cell Proliferation

Marrero

Schieffer

et al. 1997

Journal of Biological Chemistry

220

170

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In vascular smooth muscle cells, the induction of early growth response genes involves the Janus kinase (JAK)/ signal transducer and activators of transcription (STAT) and the Ras/Raf-1/mitogen-activated protein kinase cascades. In the present study, we found that electroporation of antibodies against MEK1 or ERK1 abolished vascular smooth muscle cell proliferation in response to either platelet-derived growth factor or angiotensin II. However, anti-STAT1 or -STAT3 antibody electroporation abolished proliferative responses only to angiotensin II and not to platelet-derived growth factor. AG-490, a specific inhibitor of the JAK2 tyrosine kinase, prevented proliferation of vascular smooth muscle cells, complex formation between JAK2 and Raf-1, the tyrosine phosphorylation of Raf-1, and the activation of ERK1 in response to either angiotensin II or platelet-derived growth factor. However, AG-490 had no effect on angiotensin II-or platelet-derived growth factor-induced Ras/Raf-1 complex formation. Our results indicate that: 1) STAT proteins play an essential role in angiotensin II-induced vascular smooth muscle cell proliferation, 2) JAK2 plays an essential role in the tyrosine phosphorylation of Raf-1, and 3) convergent mitogenic signaling cascades involving the cytosolic kinases JAK2, MEK1, and ERK1 mediate vascular smooth muscle cell proliferation in response to both growth factor and G protein-coupled receptors.Previous work by our laboratory (1-6) on cultured rat aortic vascular smooth muscle cells (VSMC) 1 and phenotypically similar glomerular mesangial cells has shown that protein tyrosine phosphorylation plays a critical role in angiotensin II (Ang II)-mediated intracellular signaling cascades. This is true despite the fact that G protein-coupled receptors in general and the Ang II AT 1 receptor in particular possess no intrinsic tyrosine kinase activity. It is also now recognized that Ang II can act not only as a vasoactive peptide but also as a growth factor. In particular, Ang II has been shown to stimulate proliferative and hypertrophic growth in VSMC, glomerular mesangial cells, cardiac fibroblasts, and myocytes via AT 1 receptor binding (4, 7-9). Like classic growth factors (e.g. platelet-derived growth factor (PDGF) and epidermal growth factor) and some cytokines (e.g. interferons and interleukins) (4, 8 -10), Ang II is also capable of stimulating a rapid increase in the mRNA levels of c-fos, an early growth response gene implicated in VSMC proliferation (4,7,8). However, the Ang II-stimulated intracellular signaling cascades responsible for c-fos induction and therefore proliferation in VSMC have not been well defined.One candidate mitogenic signaling cascade involves the activation of the small GTP-binding protein, Ras, which is traditionally mediated via classic growth factor receptors (4). Ras activation promotes the formation of a membrane-bound complex with Raf-1 (a serine/threonine protein kinase). Subsequent tyrosine phosphorylation of Raf-1 leads to its activation and the sequential stimulati...

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Section: Methodsmentioning

confidence: 99%

Role of Janus Kinase/Signal Transducer and Activator of Transcription and Mitogen-activated Protein Kinase Cascades in Angiotensin II- and Platelet-derived Growth Factor-induced Vascular Smooth Muscle Cell Proliferation

Marrero

Schieffer

et al. 1997

Journal of Biological Chemistry

220

170

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“…Control survival activity of Gas6 in the absence of drug was monitored in parallel experiments. After 20 h, cell viability was evaluated by measuring the uptake of 3-[4,5-dimethyltiazol-2-yl]-2,5-diphenyltetrazoliumbromide (MTT) for 4 h at 378C (Buttke et al, 1993). The results of four independent experiments are shown in the Figure 5b.…”

Section: Map Kinase Cascade Is Not Required For Gas6-dependent Survivalmentioning

confidence: 99%

Gas6-mediated survival in NIH3T3 cells activates stress signalling cascade and is independent of Ras

et al. 1999

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“…The biological activity of biotin-TPO was confirmed to be the same as unlabeled TPO by a MTS assay. 11 Platelets obtained from a single normal donor which were transfused into patient 1 and those obtained from patient 1 1 h after the platelet transfusion at the time of the nadir of platelet count as shown in Figure 1a (indicated with an arrow) were examined for TPO binding assay. Platelets from the latter were prepared from plateletrich plasma obtained from whole blood anticoagulated with ACD.…”

Section: Non-radioisotopic Tpo Binding Assay Of Plateletsmentioning

confidence: 99%

Serum thrombopoietin levels in patients correlate inversely with platelet counts during chemotherapy-induced thrombocytopenia

et al. 1998

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We studied serum thrombopoietin (TPO) levels and circulating numbers of platelet during five courses of myelosuppressive post-remission chemotherapy in three patients with acute leukemia in complete remission. Serum TPO levels were measured by a newly developed and sensitive sandwich enzyme-linked immunosorbent assay. In all courses, serum TPO levels changed reciprocally with the platelet counts. When platelets were transfused into patients near the time of platelet nadir, the TPO levels dropped temporarily, while platelet counts temporarily increased. In addition, platelets obtained after transfusion in a thrombocytopenic patient showed lower binding to biotinylated TPO than donor platelets prior to the transfusion. The finding indicated that the TPO receptors were saturated with endogenous TPO of the patient with a high serum TPO level. These results suggest that the platelet mass directly regulates serum TPO levels by receptor-mediated absorption and is one of the major regulators of serum TPO levels in humans.

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Use of an aqueous soluble tetrazolium/formazan assay to measure viability and proliferation of lymphokine-dependent cell lines

Cited by 300 publications

References 19 publications

Role of Janus Kinase/Signal Transducer and Activator of Transcription and Mitogen-activated Protein Kinase Cascades in Angiotensin II- and Platelet-derived Growth Factor-induced Vascular Smooth Muscle Cell Proliferation

Role of Janus Kinase/Signal Transducer and Activator of Transcription and Mitogen-activated Protein Kinase Cascades in Angiotensin II- and Platelet-derived Growth Factor-induced Vascular Smooth Muscle Cell Proliferation

Gas6-mediated survival in NIH3T3 cells activates stress signalling cascade and is independent of Ras

Serum thrombopoietin levels in patients correlate inversely with platelet counts during chemotherapy-induced thrombocytopenia

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