The objective of this study is to propose a more accurate and faster MTT [3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide] colorimetric assay (MCA) for quantitative measurement of polypeptide bacteriocins in solutions with nisin as an example. After an initial incubation of nisin and indicator bacterium Micrococcus luteus NCIB 8166 in tubes, MTT was added for another incubation period. After that, nisin was quantified by estimating the number of viable bacteria based on measuring the amount of purple formazan produced by cleavage of yellow tetrazolium salt MTT. Then MCA was compared to a standard agar diffusion assay (ADA). The results suggested a high correlation coefficient (r(2)=0.975+/-0.004) between optical density (OD) and the inhibitory effect of nisin on a bacterial strain Micrococcus luteus NCIB 8166 at a range of 0.125-32 IU/ml. The MCA described in this study was very quick. Quantification of nisin took only 7-8 h and the detection limit was at the level of 0.125 IU/ml when compared to 12 IU/ml and 24-28 h for ADA. The MCA provides an accurate and rapid method for quantification of nisin in solutions and is expected to be used for quantification of other antimicrobial substances.
Bacteriocins ; analysis ; metabolism ; Colorimetry ; methods ; Immunodiffusion ; Micrococcus luteus ; metabolism ; Nisin ; Regression Analysis ; Tetrazolium Salts ; analysis ; Thiazoles ; analysis ; Time Factors

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

To investigate 1alpha,25-(OH)2D3 regulation of matrix metalloproteinase-9 (MMP-9) protein expression during osteoclast formation and differentiation, receptor activator of nuclear factor kappaB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) were administered to induce the differentiation of RAW264.7 cells into osteoclasts. The cells were incubated with different concentrations of 1alpha,25-(OH)2D3 during culturing, and cell proliferation was measured using the methylthiazol tetrazolium method. Osteoclast formation was confirmed using tartrate-resistant acid phosphatase (TRAP) staining and assessing bone lacunar resorption. MMP-9 protein expression levels were measured with Western blotting. We showed that 1alpha,25-(OH)2D3 inhibited RAW264.7 cell proliferation induced by RANKL and M-CSF, increased the numbers of TRAP-positive osteoclasts and their nuclei, enhanced osteoclast bone resorption, and promoted MMP-9 protein expression in a concentration-dependent manner. These findings indicate that 1alpha,25-(OH)2D3 administered at a physiological relevant concentration promoted osteoclast formation and could regulate osteoclast bone metabolism by increasing MMP-9 protein expression during osteoclast differentiation.
Acid Phosphatase/metabolism ; Animals ; Blotting, Western ; Calcitriol/*pharmacology ; Calcium Channel Agonists/pharmacology ; *Cell Differentiation ; Cell Line ; Cell Proliferation ; Gene Expression Regulation, Enzymologic/*drug effects ; Isoenzymes/metabolism ; Matrix Metalloproteinase 9/*genetics/metabolism ; Mice ; Osteoclasts/*cytology/*enzymology ; Tetrazolium Salts ; Thiazoles

PURPOSE: The purpose of this study is to investigate the mechanism of cellular proliferation of electromagnetic field (EMF) on human intervertebral disc (IVD) cells. MATERIALS AND METHODS: Human IVD cells were cultured three-dimensionally in alginate beads. EMF was exposed to IVD cells with 650Omega, 1.8 millitesla magnetic flux density, 60 Hz sinusoidal wave. Cultures were divided into a control and EMF group. Cytotoxicity, DNA synthesis and proteoglycan synthesis were measured by MTT assay, [3H]-thymidine, and [35S]-sulfate incorporation. To detect phenotypical expression, reverse transcription-polymerase chain reactions (RT-PCR) were performed for aggrecan, collagen type I, and type II mRNA expression. To assess action mechanism of EMF, IVD cells were exposed to EMF with NG-Monomethyl-L-arginine (NMMA) and acetylsalicylic acid (ASA). RESULTS: There was no cytotoxicity in IVD cells with the EMF group in MTT assay. Cellular proliferation was observed in the EMF group (p < 0.05). There was no difference in newly synthesized proteoglycan normalized by DNA synthesis between the EMF group and the control. Cultures with EMF showed no significant change in the expression of aggrecan, type I, and type II collagen mRNA compared to the control group. Cultures with NMMA (blocker of nitric oxide) or ASA (blocker of prostaglandin E2) exposed to EMF demonstrated decreased DNA synthesis compared to control cultures without NMMA or ASA (p < 0.05). CONCLUSION: EMF stimulated DNA synthesis in human IVD cells while no significant effect on proteoglycan synthesis and chondrogenic phenotype expressions. DNA synthesis was partially mediated by nitric oxide and prostaglandin E2. EMF can be utilized to stimulate proliferation of IVD cells, which may provide efficient cell amplification in cell therapy to degenerative disc disease.
Adult ; Aspirin/pharmacology ; Cell Proliferation/*radiation effects ; Collagen/metabolism ; Dinoprostone/metabolism ; *Electromagnetic Fields ; Enzyme Inhibitors/pharmacology ; Female ; Humans ; Intervertebral Disk/*pathology/radiation effects ; Male ; Middle Aged ; Nitric Oxide/metabolism ; Tetrazolium Salts/pharmacology ; Thiazoles/pharmacology ; omega-N-Methylarginine/pharmacology

As the incidence of nontuberculous mycobacterial infection has been increasing recently in Korea, the importance of drug susceptibility test for clinical isolates of mycobacteria has become larger. In this study we determined the antimicrobial susceptibility patterns of clinical isolates of M. fortuitum and M. abscessus in Korea, and evaluated the efficacy of a modified broth microdilution method using 2,3-diphenyl-5-thienyl-(2)-tetrazolium chloride (STC), in terms of its ability to provide accurate and easy-to-read minimal inhibitory concentration (MIC) endpoints for the susceptibility testing of rapidly growing mycobacteria. Most isolates of M. fortuitum and M. abscessus in Korea are susceptible or intermediately susceptible to amikacin, cefoxitin, ciprofloxacin, and clarithromycin. Many isolates of M. fortuitum are susceptible to doxycycline, sulfamethoxazole, and imipenem, while many M. abscessus isolates are resistant to these drugs. In the present study, the modified broth microdilution method using STC was found to be reliable, easy to read, and inexpensive for M. fortuitum and M. abscessus susceptibility testing. The modified colorimetric MIC testing method using STC was proven to be a useful surrogate for RGM antibiotic susceptibility testing.
Anti-Bacterial Agents/pharmacology ; Cefoxitin/pharmacology ; Chemistry, Pharmaceutical/methods ; Ciprofloxacin/pharmacology ; Clarithromycin/pharmacology ; Colorimetry/methods ; Drug Resistance, Bacterial ; Korea ; *Microbial Sensitivity Tests ; Mycobacterium/*metabolism ; Mycobacterium fortuitum/*metabolism ; Tetrazolium Salts/*pharmacology

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 evaluate the effect of quorum sensing luxS gene on biofilm formation through construction of a luxS overexpression strain by Streptococcus mutans (Sm).

METHODSIn order to construct pIB-luxS plasmid, the luxS gene fragment amplified by PCR was inserted into the shuttle plasmid pIB169 by corresponding double digests. The pIB-luxS plasmid was linearized electro-transformed into Sm cell and the overexpression strain was selected on chloramphenicol plate and testified by electrophoresis and western blot. The growth rate of both Sm wild type strain and its luxS overexpression strain were observed. Methyl thiazolyl tetrazolium (MTT) assay method was used to compare the biofilm formation quantification by both strains at different time points and containing different sucrose. The structures of the biofilms were observed by using confocal laser scanning microscopy, and biofilm-related gene expressions were investigated by real-time PCR. All experiments were performed in triplicate.

RESULTSThe luxS overexpression strain was successfully constructed and confirmed by electrophoresis and Western blotting. The planktonic growth mode of the wild-type and luxS overexpression strain showed no difference, but biofilm formed by Sm overexpression strain was 0.400 ± 0.009 and 0.609 ± 0.041 at 14 and 24 h, higher than the wild type strain biofilm at the same time point (0.352 ± 0.028 and 0.533 ± 0.014, respectively, P < 0.05). After adding 0.125% sucrose, biofilm formed by Sm overexpression strain raised to 1.041 ± 0.038, higher than that by the wild type strain (0.831 ± 0.020, P < 0.05). The biofilm formed by both strains were also increased with the sucrose concentration increase, but there was no difference between them. The overexpression strain aggregated into distinct clusters on structure, genes expression including gtfB, ftf, gbpB, relA, brpA, smu630, comDE, vicR were increased (6.10 ± 0.12, 3.34 ± 0.07, 8.75 ± 0.13, 2.96 ± 0.04, 5.20 ± 0.19, 2.20 ± 0.06, 2.32 ± 0.07 and 10.67 ± 0.57 fold) compared to the wild-type strain (P < 0.05).

CONCLUSIONSQuorum sensing luxS gene can promote the biofilm formation of Sm.

Bacterial Proteins ; genetics ; metabolism ; Biofilms ; growth & development ; Carbon-Sulfur Lyases ; genetics ; metabolism ; Microscopy, Confocal ; Plasmids ; genetics ; Quorum Sensing ; genetics ; Real-Time Polymerase Chain Reaction ; Streptococcus mutans ; physiology ; Tetrazolium Salts ; Time Factors

OBJECTIVETo examine the expression of heat shock protein 25 (HSP-25) in dental rat follicles in vivo and in vitro in order to investigate the possible effect of HSP-25 on cell proliferation and alkaline phosphatase (ALP) activity.

METHODSThe expression of HSP-25 in mandibles of postnatal rats from day 1, 3, 5, 7, 9, 11 was examined by immunohistochemistry in vitro, the expression of HSP-25 in the dental follicle cells was detected by the indirect immunofluorescence method. Methyl thiazolyl tetrazolium (MTT) assay, flowcytometry and ALP assay were used to detect the effect of HSP-25 on rat dental follicles.

RESULTSHSP-25 expression was absent or weak in rat dental follicle cells at early postnatal stage and present from day 5 till day 11. HSP-25 was detected in the cytoplasm of cultured dental follicle cells. MTT results showed no effect could be detected on dental follicle cell proliferation after stimulation of different concentrations of HSP-25 on day 1, 2, 3. Flowcytometry results also exhibited no difference in cell cycles after stimulation of HSP-25 at 0 microg/L and 100 microg/L. HSP-25 at a concentration of 50 microg/L and 100 microg/L could up-regulate the ALP activity on day 9.

CONCLUSIONSExpression of HSP-25 increases chronologically in the rat dental follicle cells. HSP-25 locates in the cytoplasm of cultured rat dental follicle cells. HSP-25 has no effect on the proliferation of dental follicle cells, however it can up-regulate the ALP activity.

Alkaline Phosphatase ; metabolism ; Animals ; Cell Proliferation ; Cells, Cultured ; Dental Sac ; metabolism ; Flow Cytometry ; Fluorescent Antibody Technique, Indirect ; HSP27 Heat-Shock Proteins ; physiology ; Immunohistochemistry ; Rats ; Tetrazolium Salts ; Thiazoles ; Up-Regulation