In order to establish a new more rapid, safe and sensitive colorimetric assay for the proliferation of leukemic cells, MTS/pms has been developed. This automated colorimetric assay is based on the characteristic of viable and metabolically active leukemic cells to cleave MTS/pms into a water-soluble product whose optical density is determined at 492 nm by an automated microtiter-plate reader photometer. The results indicated that only active leukemic cells cleaved MTS/pms into product measured, and dead cells did not reduce MTS/pms. A linear relations hip were found between the viable cell number and optical density of MTS/pms cleaved by HL-60 and K562 cell (r = 0.963). Compared with MTT and INT assays, the reduced product of MTS/pms is water-soluble. It is concluded that MTS/pms colorimetric assay is more rapid, accurate and sensitive for the bioassay of proliferation of leukemic cells.
Cell Division ; Colorimetry ; methods ; Formazans ; metabolism ; HL-60 Cells ; Humans ; K562 Cells ; Leukemia ; pathology ; Methylphenazonium Methosulfate ; metabolism ; Tetrazolium Salts ; metabolism ; Thiazoles ; metabolism

The methanol extract from the leaves of Petasites japonicus Maxim (PJ) was studied for its (anti-)mutagenic effect with the SOS chromotest and reverse mutation assay. The (anti-)carcinogenic effects were evaluated by the cytotoxicity on human cancer line cells and by the function and the expression of gap junctions in rat liver epithelial cell. PJ extracts significantly decreased spontaneous beta-galactosidase activity and beta-galactosidase activity induced by a mutagen, ICR, in Salmonella (S.) typhimurium TA 1535/pSK 1002. All doses of the extract (0.08-100 mg/plate) decreased the reversion frequency induced by benzo (alpha)pyrene (BaP) in S. typhimurium TA 98. It decreased not only the spontaneous reversion frequency but also that induced by BaP in S. typhimurium TA 100. PJ extract showed greater cytotoxic effects on human stomach, colon and uterus cancer cells than on other cancer cell types and normal rat liver epithelial cells. Dye transfers though gap junctions were significantly increased by PJ extracts at concentrations greater than 200 microg/mL and the inhibition of dye transfer by 12-O-tetradecanoylphorobol-13-acetate (TPA) was obstructed in all concentrations of PJ. PJ significantly increased the numbers of gap junction protein connexin 43, and increased the protein expression decreased by TPA in a dose-dependent manner. Based on these findings, PJ is suggested to contain antimutagenic and anticarcionogenic compounds.
Animals ; Cell Line, Tumor ; Cell Survival/*drug effects ; Formazans/chemistry ; Gap Junctions/*metabolism ; Humans ; Mutagenicity Tests ; Petasites/*metabolism ; Plant Extracts/*pharmacology ; Plant Leaves/metabolism ; Rats ; Tetrazolium Salts/chemistry

OBJECTIVETo determine the effects of organic amine diphenhydramine on the 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide dye (MTT) reduction assay.

METHODSThe primarily cultured cortical astrocytes were incubated with various concentrations of diphenhydramine for 24 h. To analyze the effects of diphenhydramine and other organic amines on the MTT assay, the data obtained from the MTT assay were compared with the results obtained from morphological observation and hoechst 33342 and propidium iodide (PI) nucleus double staining.

RESULTThe MTT assay showed that diphenhydramine (10(-4)mol/L), pyrilamine (10 (-4)mol/L) and zolantidine (10 (-5)mol/L) caused a significant increase in MTT reduction in astrocytes. However there was no proliferation, apoptosis or necrosis detected by hoechst and PI nucleus double staining. Light microscopy revealed that exocytosis of formazan granules was inhibited by diphenhydramine.

CONCLUSIONDiphenhydramine and other organic amines may enhance MTT reduction by suppression of MTT formazan exocytosis in astrocytes, which may affect the results of cell viability studies.

Animals ; Astrocytes ; drug effects ; metabolism ; physiology ; Cell Survival ; Cells, Cultured ; Diphenhydramine ; pharmacology ; Drug Interactions ; Formazans ; pharmacokinetics ; Rats ; Rats, Sprague-Dawley ; Tetrazolium Salts ; pharmacokinetics

OBJECTIVETo establish a simpler and more accurate method for evaluating in vitro ischemic injury and neuroprotective effects of drugs through improving experimental instrument and quantitative index in mouse brain slices.

METHODSAn incubation instrument was developed and its application tested. 2,3,5-triphenyltetrazolium chloride (TTC) was used as a substrate to biosynthesize formazan standard in mouse brain slices, and formazan was isolated, purified and identified. Ischemic injury of mouse brain slices was induced by oxygen/glucose deprivation (OGD), the produced formazan from TTC in the cortex and striatum was measured at 490 nm spectrophotometrically. Edaravone and ONO-1078 were added into the incubation medium to observe their neuroprotective effects.

RESULTThe incubation instrument worked well for incubating brain slices and obtaining stable results efficiently. Standard formazan was biosynthesized and purified with a purity of 99.3%, and showed a linear range of 0.05 - 1 mg/ml in absorbance at 490 nm (r=0.9997). OGD decreased formazan production in the cortex and striatum in a duration-dependent manner. Edaravone (0.01 to 1 micromol/L) recovered OGD-induced decrease of formazan production, but ONO-1078 showed no effect.

CONCLUSIONThe incubation instrument and quantitative measurement of formazan developed in this study are efficient,accurate and simple for evaluating ischemic injury and neuroprotection,which can be used in screening of neuroprotective drugs in vitro.

Alprostadil ; analogs & derivatives ; pharmacology ; Animals ; Antipyrine ; analogs & derivatives ; pharmacology ; Brain Ischemia ; diagnosis ; drug therapy ; Formazans ; metabolism ; Male ; Mice ; Mice, Inbred ICR ; Neuroprotective Agents ; pharmacology ; Staining and Labeling ; Tetrazolium Salts ; metabolism

OBJECTIVETo investigate the role of mitochondrial calcium uniporter in cardioprotection elicited by ischemic postconditioning (Postcond).

METHODSMale Sprague-Dawley rats were used for Langendorff isolated heart perfusion. The hearts subjected to global ischemia for 30 min followed by 120 min of reperfusion. Left ventricular developed pressure (LVDP), maximal rise/fall rate of left ventricular pressure (± dP/dtmax) were measured. The level of lactate dehydrogenase (LDH) in the coronary effluent was measured spectrophotometrically, the content of formazan of myocardium was also measured at the end of reperfusion.

RESULTCompared to I/R group, Postcond had an significant increase in the mechanical function of the left ventricle, with LDH release reduced and the content of formazan increased. Spermine, the opener of mitochondrial calcium uniporter, deteriorated the mechanical function of left ventricle and decreased the formazan content, and increased LDH release. Ruthenium red, the inhibitor of mitochondrial calcium uniporter, increased the mechanical function of the left ventricle, decreased the LDH release, but the content of formazan was not increased.

CONCLUSIONThe inhibition of mitochondrial calcium uniporter is involved in the mechanisms of ischemic postconditioning.

Animals ; Calcium Channels ; metabolism ; physiology ; Disease Models, Animal ; Formazans ; analysis ; Heart ; physiopathology ; Ischemic Postconditioning ; L-Lactate Dehydrogenase ; analysis ; Male ; Myocardial Reperfusion Injury ; metabolism ; physiopathology ; prevention & control ; Rats ; Rats, Sprague-Dawley

AIMTo determine the effect of histamine on ischemia-induced cellular edema and viability reduction in rat hippocampal slices, and the involved subtypes of histamine receptor in this effect.

METHODSIn vitro ischemic injury of hippocampal slices was induced by oxygen-glucose deprivation (OGD). The slice injury was determined by real-timely measuring the changes of light transmittance (LT) for the cellular edema in CA1 region of the hippocampal slice, and by detecting the product of 2, 3, 5-triphenyltetrazolium chloride (TTC), formazan, for the slice viability. The effect of histamine at various concentrations on the slice injury was observed, and the blockage by antagonists of histamine receptors was also investigated.

RESULTSHistamine (0.01-10 micromol x L(-1)) inhibited the peak value of LT during OGD in hippocampal slices and improved the reduced viability after OGD. Diphenhydramine (0.1-10 micromol x L(-1)), an H1 receptor antagonist, did not affect the effect of histamine, while cimetidine (0.1-10 micromol x L(-1)), an H2 receptor antagonist, partly abolished the protective effect of histamine.

CONCLUSIONHistamine protects hippocampal slices against ischemia-induced cellular edema and viability reduction; this effect might be mediated via, at least partly, H2 receptor.

Animals ; Cell Hypoxia ; Cell Survival ; drug effects ; Cimetidine ; pharmacology ; Diphenhydramine ; pharmacology ; Formazans ; metabolism ; Glucose ; deficiency ; Hippocampus ; drug effects ; metabolism ; pathology ; Histamine ; pharmacology ; Histamine H1 Antagonists ; pharmacology ; Histamine H2 Antagonists ; pharmacology ; Male ; Neuroprotective Agents ; pharmacology ; Rats ; Rats, Sprague-Dawley

Amyloid beta (Abeta) neurotoxicity is believed to play a critical role in the pathogenesis of Alzheimer's disease (AD) mainly because of its deposition in AD brain and its neuronal toxicity. However, there have been discrepancies in Abeta-induced cytotoxicity studies, depending on the assay methods. Comparative analysis of Abeta42-induced in vitro cytotoxicity might be useful to elucidate the etiological role of Abeta in the pathogenesis of AD. In this study, MTT, CCK-8, calcein-AM/EthD-1 assays as well as thorough microscopic examinations were comparatively performed after Abeta42 treatment in a neuronal precursor cells (NT2) and a somatic cells (EcR293). Extensive formation of vacuoles was observed at the very early stage of Abeta42 treatment in both cells. Early observation of Abeta42 toxicity as seen in vacuole formation was also shown in MTT assay, but not in CCK-8 and calcein-AM/EthD-1 assays. In addition, Abeta42 treatment dramatically accelerated MTT formazan exocytosis, implying its effect on the extensive formation of cytoplasmic vacuoles. Abeta42 seems to cause indirect inhibition on the intracellular MTT reduction as well as vacuole formation and exocytosis enhancement. Following the acute cellular dysfunction induced by Abeta42, the prolonged treatment of micromolar concentration of Abeta42 resulted in slight inhibition on redox and esterase activity. The early Abeta42-induced vacuolated morphology and later chronic cytotoxic effect in neuronal cell might be linked to the chronic neurodegeneration caused by the accumulation of Abeta42 in AD patients' brain.
Amyloid beta-Protein/*toxicity ; Animals ; Cell Death/drug effects ; Cell Line ; Dose-Response Relationship, Drug ; Exocytosis/*drug effects ; Formazans ; Neurons/*drug effects/metabolism/*pathology ; Peptide Fragments/*toxicity ; Research Support, Non-U.S. Gov't ; Tetrazolium Salts ; Time Factors ; Vacuoles/*drug effects

OBJECTIVETo determine the effect of aspirin on cell proliferation, alkaline phosphatase (ALP) activity, cell cycle and apoptosis in murine bone marrow stromal cells, so as to explore an appropriate dose range to improve bone regeneration in periodontal treatment.

METHODSST2 cells were stimulated with aspirin (concentrations of 1, 10, 100 and 1 000 μmol/L) for 1, 2, 3, 5 and 7 d. Cell proliferation was measured by methyl thiazolyl tetrazolium (MTT) assay. After ST2 cells were treated for 1, 3 and 7 d, ALP activity was measured by ALP kit, cell cycle and apoptosis were measured by flow cytometry (FCM) after treated for 48 h.

RESULTSMTT assays showed that various doses of aspirin have different effects on the cell growth. Briefly, lower concentrations (1, 10 μmol/L) of aspirin promoted the cell growth, the A value of 0, 1 and 10 μmol/L aspirin 7-day-treated cells were 0.313±0.012, 0.413±0.010 and 0.387±0.017 respectively (P <0.01 vs control), and so did the ALP level ([4.3±0.9], [6.0±0.3] and [7.7±0.4] μmol·min(-1)·g(-1), P <0.05 vs control), while higher concentrations, especially 1000 μmol/L of aspirin might inhibit the cell growth with time going, A value and ALP level were 0.267±0.016, (4.3±1.3) μmol·min(-1)·g(-1) respectively (P <0.05 vs control). Cell cycle analysis revealed no changes in comparison to control cells after treatment with 1 or 10 μmol/L aspirin, but it was observed that cell mitosis from S phase to G2/M phase proceeded at higher concentrations of 100 μmol/L aspirin, and the cell cycle in phase G0/G1 arrested at 1000 μmol/L. Parallel apoptosis/necrosis studies showed that the percentage of cells in apoptosis decreased dramatically at all doses of aspirin, the apoptosis rates of ST2 cells responded to 0, 1, 10, 100 and 1000 μmol/L aspirin were (11.50±0.90)%, (5.30±0.10)%, (5.50±0.10)%, (4.90±0.90)% and (7.95±0.25)% respectively (P<0.05 vs control).

CONCLUSIONSThis study demonstrated that lower dosage of aspirin can promote ST2 cells growth, osteogenic activity and inhibit its apoptosis. Aspirin maybe used for the bone reconstruction with a proper concentration.

Alkaline Phosphatase ; metabolism ; Animals ; Apoptosis ; drug effects ; Aspirin ; administration & dosage ; pharmacology ; Bone Regeneration ; Cell Cycle ; drug effects ; Cell Division ; Cell Line, Tumor ; Cell Proliferation ; Flow Cytometry ; Formazans ; Mesenchymal Stromal Cells ; cytology ; drug effects ; enzymology ; Mice ; Periodontics ; Tetrazolium Salts ; Time Factors

Taxol is a clinically useful anticancer drug against a variety of cancers. Although it has been known that taxol induces the apoptosis of cancer cells through cytochrome C release and the activation of caspases, the effect of taxol on dendritic cells (DCs) has not been studied. In this study, taxol enhanced the expression of MHC class II on DCs, compared to medium-treated immature DCs. Surprisingly, the viability of DCs was not decreased by taxol, whereas that of cancer cells was. It was confirmed that taxol did not induce the apoptosis of DCs based on annexin V-FITC/propidium iodide (PI) staining assay. Since previous study demonstrated that taxol induced the production of nitric oxide (NO) related to the viability of DCs, the level of NO from taxol-treated DCs was determined. Any significant amount of NO was not detected. Although taxol enhanced the expression of a maturation marker, MHC class II molecules, it strikingly inhibited the proliferation of splenic T lymphocytes activated by DCs. Taken together, this study demonstrated that taxol induced an altered maturation of DCs, the increase of MHC class II molecule but the inhibition of proliferation of splenic T lymphocytes. It is suggested that taxol may induce the immunosuppression in patients with cancer by the inhibition of DC-activated T cell proliferation, but not by the direct killing of DCs.
Animals ; Antineoplastic Agents, Phytogenic/*pharmacology ; Apoptosis/drug effects ; Coloring Agents/metabolism ; Dendritic Cells/cytology/*drug effects/immunology ; Female ; Flow Cytometry ; Formazans/metabolism ; Histocompatibility Antigens Class II/biosynthesis ; Lymphocyte Activation/drug effects ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Nitric Oxide/metabolism ; Paclitaxel/*pharmacology ; T-Lymphocytes/cytology/immunology/metabolism ; Tetrazolium Salts/metabolism

A normal prion protein (PrPc) is converted to a proteaseresistant isoform by an apparent self-propagating activity in transmissible spongiform encephalopathy, a neurodegenerative disease. The cDNA encoding open reading frame (ORF) of the bovine prion protein gene (Prnp) was cloned from Korean cattle by PCR, and was transfected into Chinese hamster ovary (CHO-K1) cells using lipofectamine. The gene expression of the cloned cDNA was confirmed by RT-PCR and Western blotting with the monoclonal antibody, 6H4. Cellular changes in the transfected CHO-K1 cells were investigated using parameters such as MTT, lactate dehydrogenase (LDH), and superoxide dismutase (SOD) activities, as well as nitric oxide (NO) production, and an apoptosis assay. In the MTT and LDH assays, the bovine PrnP-transfectant showed a lower proliferation rate than the wild-type (p < 0.05). Production of NO, after LPS or ConA stimulation, was not detected in either transfectants or CHO-K1 cells. In SOD assay under ConA stimulation, the SOD activity of transfectants was 10 times higher than that of CHO-K1 cells at 6 h after treatment (p < 0.05). The genomic DNA of both the transfectants and control cells began to be fragmented at 6 h after treatment with cyclohexamide. Caspase-3 activity was reduced by transfection with the bovine Prnp (p < 0.05). Conclusively, the viability of transfectants expressing exogenous bovine Prnp was decreased while the capacities for cellular protection against antioxidative stress and apoptosis were increased.
Animals ; Apoptosis/physiology ; CHO Cells/cytology/enzymology/*physiology ; Caspase 3/metabolism ; Cattle ; Cell Growth Processes/physiology ; Cloning, Molecular ; Cricetinae ; Cricetulus ; Encephalopathy, Bovine Spongiform/genetics/*pathology ; Formazans ; Hydro-Lyases/metabolism ; Nitric Oxide/metabolism ; Prions/biosynthesis/genetics/*physiology ; Superoxide Dismutase/metabolism ; Tetrazolium Salts ; Transfection