• Title, Summary, Keyword: MTT

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Development of Sperm MTT Assay for Its Application in Boar Semen

  • Jang, Hyun-Yong;Lee, Hee-Young;Cheong, Hee-Tae;Kim, Jong-Taek;Park, In-Chul;Park, Choon-Keun;Yang, Boo-Keun
    • Journal of Embryo Transfer
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    • v.25 no.4
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    • pp.229-235
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    • 2010
  • The MTT assay is one of superior evaluation methods widely used to analyze the viability of metabolically active cell. It can be used to determine the percentage of viable sperm through measurement of the reduction of MTT granules at mitochondria in sperm tail. The purpose of this study is to determine the optimal condition of a simple and easy MTT assay to validate boar sperm viability and compare the accuracy of this test with microscopic examination. The MTT reduction rate for sperm viability were analyzed in microtiter plates (96 well) from 1 hr to 5 hr incubation periods at $37^{\circ}C$ using spectrophotometer (microplate reader) at 550 nm wavelength. The remainder of semen sample was simultaneously examined to compare the correlation of accuracy between MTT assay and other sperm parameters. Those sperm parameters were included the motility, survival rates, membrane integrity, mitochondria activity and acrosome integrity. The OD values of MTT assay (MTT reduction rates) did not greatly change at 1 hr to 5 hr incubation periods in different proportion of live and freeze-killed sperms (dead sperm). The MTT reduction rates or survival rates were decreased according to the different concentration of live and dead sperm. The linear regression at 1 hr and 4 hr incubation periods in sperm MTT assay was y=291.55x-72.176 and y= 180.64x-44.569, respectively. There are high correlation between 1 hr and 4 hr incubation periods (p<0.001). The results of MTT assay and other sperm parameters has a positive correlation (p<0.01 or 0.05). The correlation coefficients for MTT assay was 0.88115 for motility, 0.89868 for survival rates, 0.91722 for membrane integrity and 0.77372 for acrosome integrity, respectively. In conclusion, the MTT assay can be used as a reliable and efficient evaluation method for boar sperm viability. It can be use practical means to evaluate the quality of boar sperm by a fast, inexpensive and easy method.

Effects of various lights, solvents, and zinc protoporphyrin on the chemical behavior of MTT formazan (빛, 용매와 zinc protoporphyrin에 의한 MTT 포마잔의 화학적 동태 변화)

  • Kim, Joo Hyoun;Hong, Jungil
    • Korean Journal of Food Science and Technology
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    • v.50 no.1
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    • pp.1-7
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    • 2018
  • The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay is commonly used for analyzing the cell viability. In this study, effects of various solvents, different lights, and zinc protoporphyrin (ZnPP) on the chemical behavior of MTT formazan were investigated. The color response of MTT formazan in NaOH was highly pronounced; the absorbance of MTT formazan in 0.1 N NaOH at 550 nm was >2-fold higher than that in water, dimethyl sulfoxide (DMSO), methanol, and ethanol. MTT formazan in DMSO and NaOH (>0.1 N) was relatively stable under fluorescent and UV light at 365 nm; its rapid degradation was induced under UV light at 254 nm in all solvents. ZnPP degraded MTT formazan under light in a time- and concentration-dependent manner; MTT formazan in 0.1 N NaOH was the most sensitive to ZnPP, followed by DMSO. These results suggest that NaOH and DMSO might be suitable media for MTT formazan for monitoring photosensitizing properties.

The Optimal Condition of Performing MTT Assay for the Determination of Radiation Sensitivity (방사선 감수성 측정법으로서 MTT 법 시행 시의 최적 조건에 대한 연구)

  • Hong, Se-Mie;Kim, Il-Han
    • Radiation Oncology Journal
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    • v.19 no.2
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    • pp.163-170
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    • 2001
  • Purpose : The measurement of radiation survival using a clonogenic assay, the established standard, can be difficult and time consuming. In this study, We have used the MTT assay, based on the reduction of a tetrazolium salt to a purple formazan precipitate by living cells, as a substitution for clonogenic assay and have examined the optimal condition for performing this assay in determination of radiation sensitivity. Materials and Methods : Four human cancer cell lines - PCI-1, SNU-1066, NCI-H630 and RKO cells have been used. For each cell line, a clonogenic assay and a MTT assay using Premix WST-1 solution, which is one of the tetrazolium salts and does not require washing or solubilization of the precipitate were carried out after irradiation of 0, 2, 4, 6, 8, 10 Gy. For clonogenic assay, cells in $25\;cm^2$ flasks were irradiated after overnight incubation and the resultant colonies containing more than 50 cells were scored after culturing the cells for $10\~14$ days. For MTT assay, the relationship between absorbance and cell number, optimal seeding cell number, and optimal timing of assay was determined. Then, MTT assay was performed when the irradiated cells had regained exponential growth or when the non-irradiated cells had undergone four or more doubling times. Results : There was minimal variation in the values gained from these two methods with the standard deviation generally less than $5\%$, and there were no statistically significant differences between two methods according to t-test in low radiation dose (below 6 Gy). The regression analyses showed high linear correlation with the $R^2$ value of $0.975\~0.992$ between data from the two different methods. The optimal cell numbers for MTT assay were found to be dependent on plating efficiency of used cell line. Less than 300 cells/well were appropriate for cells with high plating efficiency (more than $30\%$). For cells with low plating efficiency (less than $30\%$), 500 cells/well or more were appropriate for assay. The optimal time for MTT assay was after 6 doubling times for the results compatible with those of clonogenic assay, at least after 4 doubling times was required for valid results. In consideration of practical limits of assay (12 days, in this study) cells with doubling time more than 3 days were inappropriate for application. Conclusion : In conclusion, it is found that MTT assay can successfully replace clonogenic assay of tested cancer cell lines after irradiation only if MTT assay was undertaken with optimal assay conditions that included plating efficiency of each cell line and doubling time at least.

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Synthesis and Catalytic Performance of MTT Zeolites with Different Particle Size and Acidity (다양한 입자크기와 산성도를 지닌 MTT 제올라이트의 합성 및 촉매특성 연구)

  • Park, Sung Jun;Jang, Hoi-Gu;Cho, Sung June
    • Korean Chemical Engineering Research
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    • v.56 no.4
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    • pp.600-606
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    • 2018
  • The influence of acidity in MTT zeolite of different Si/Al molar ratio's on the catalyst activity in methanol-to-olefin (MTO) reaction has been investigated. The Si/Al ratio was controlled with the Al content in the gel when N,N,N',N'-tetramethyl-1,3-diaminopropane was used as a structure directing agent (SDA). The gel composition was controlled to $20SiO_2$ : 30SDA : x (=0.25~1.25)$NaAlO_2$ : 2NaOH : $624H_2O$, which was subject to the hydrothermal synthesis at 433 K for 4 days. As the composition of sodium aluminate decreased, the particle size of MTT zeolite increased, and also the amount of acid sites decreased. To investigate the catalytic performance, MTO reaction was carried out at 673 K with $1.2h^{-1}$ WHSV. It was found that the H-MTT (1.00Al) catalyst with a Si/Al molar ratio of 24 maintained the methanol conversion over 90% for 900 min.

The Use of MTT Assay, In Vitro and Ex Vivo, to Predict the Radiosensitivity of Colorectal Cancer (In-vitro와 Ex-vivo MTT Assay를 통한 직장암의 방사선치료 감수성 예측 가능성 검증)

  • Kim, Ji-Eun;Kim, Mi-Sook;Kang, Chang-Mo;Kim, Jong-Il;Shin, Hye-Kyung;Choi, Chul-Won;Seo, Young-Seok;Ji, Young-Hoon
    • Radiation Oncology Journal
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    • v.26 no.3
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    • pp.166-172
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    • 2008
  • Purpose: The measurement of radiosensitivity of individuals is useful in radiation therapy. Unfortunately, the measurement of radiation survival using a clonogenic assay, which is the established standard, can be difficult and time consuming. The aim of this study is to compare radiosensitivity results obtained from the MTT and clonogenic assays, and to evaluate whether the MTT assay can be used on clinical specimens. Materials and Methods: HCT-8, LoVo, CT-26, and WiDr were the colon cancer cell lines used for this study. The clonogenic assay was performed to obtain the cell survival curves and surviving fractions at a dose of 2 Gy ($SF_2$) as the standard technique for radiosensitivity. Also, the MTT assay was performed for each of the cell lines (in vitro). To simulate clinical specimens, the cell lines were inoculated into nude mice, removed when the tumors reached 1 cm in diameter, and chopped. Next, the tumors were subjected to the same process involved with the MTT assay in vitro. The inhibition rates (IR) of 10 Gy or 20 Gy of irradiation for in vitro and ex vivo were calculated based on the optical density of the MTT assay, respectively. Results: According to $SF_2$ and the cell survival curve, the HCT-8 and WiDr cell lines were more resistant to radiation than LoVo and CT-26 (p<0.05). The IR was measured by in vitro. The MTT assay IR was 17.3%, 21%, 30% and 56.5% for the WiDr, HCT-8, LoVo and CT-26 cell lines, respectively. In addition, the IR measured ex vivo by the MTT assay was 23.5%, 26%, 38% and 53% in the HCT-8, WiDr, LoVo and CT-26 tumors, respectively. Conclusion: The radiosensitivity measured by the MTT assay was correlated with the measures obtained from the clonogenic assay. This result highlights the possibility that the MTT assay could be used in clinical specimens for individual radiosensitivity assays.

A Study on the Operation Frequencies of the Multiple Tie Tamper (Multiple Tie Tamper의 투입주기평가에 관한 연구)

  • 오지택
    • Proceedings of the KSR Conference
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    • pp.434-441
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    • 2000
  • This paper estabilsh the systematical scheme that evaluates the operation frequencies of the MTT(Multiple Tie Tamper). An evaluation of the operation frequencies, covering 4 different permanent ways that are Kyungbu, Homan, Jungang and Youngdong, has been carried out using real track irregularities. The deterioration rate of track irregularities used to evaluate rational operation frequencies of MTT in a block of railway track. Furthermore, this paper provides the scheme that prevents damage due to excess using of MTT and to promote efficiency of MTT application.

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Cytotoxicity of Crude Extracts of Rheum uudulatum L. with Human Kidney Epithelial Cell A498 (신장 상피세포주 A498을 이용한 대황(Rheum undulatum L.)추출물의 세포독성)

  • 나명석;진종언;조남철
    • The Korean Journal of Food And Nutrition
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    • v.13 no.5
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    • pp.460-464
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    • 2000
  • We have evaluated cytotoxic effects of four crude extracts of methylene chloride, ethyl acetate, butanol, water layer isolated Rheum undulatum in A498 cell line, human kidney epithelial cells. The cytotoxic evalutation was measured by colorimetric assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide(MTT) , neutral red(NR) and sulforhodamine protein B(SRB). These results obtained are as follows : MTT, NR and SRB quantities were significantly decreased in cultured A498 cells treated four crude extracts by increased concentrations. The cell cytotoxic effect of crude extracts of butanol layer was more stronger than others layer. The values of MTT$\sub$50/, NR$\sub$50/, SRB$\sub$50/ of crude extract of butanol layer and were measured both 0.63 mg/ml, 0.65 mg/ml, and 0.68 mg/ml, respectively and the values of water layer were 0.84 mg/ml, 0.82 mg/ml. and 0.80 mg/ml. respectively in cultured A498 cell line.

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MTT Assay Using Primary Cultured Rat Hepatocytes (백서의 초대 배양 간세포를 이용한 MTT assay$^{1)}$)

  • Ha, Hun;Yoon, Soo-Hong;Fujii, Takeru;Hori, Hitoshi
    • Environmental health and toxicology
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    • v.9 no.1_2
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    • pp.19-23
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    • 1994
  • The tetrazolium dye, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), is reduced by live but not dead cell, and this reaction is used as the end point in a rapid drug screening assay. It can also be used for accurate determinations of drug sensitivity but only if a quantative relationship is established between cell number and MTT-formazan production. Several conditions were examined to devise an in vitro assay method in primary cultured hepatocytes, such as optimum wavelength, optimal MTT concentration, optimal incubation time, and cell density.

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A New Method for Antimicrobial Susceptibility Testing of Vitro-cultured Bacteria by Means of Resonance Light Scattering Technique

  • Shi, Yu-Jun;Chen, Jun;Xu, Ming
    • Journal of Microbiology and Biotechnology
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    • v.18 no.1
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    • pp.118-123
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    • 2008
  • A new method for antimicrobial susceptibility testing of vitro-cultured bacteria on an ordinary fluorescence spectrometer was developed. The viable bacteria reduced 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to produce insoluble particles that displayed intense resonance scattering light. The assay showed a linear relationship between the number of viable bacteria and the intensity of resonance scattering light. Dead bacteria were unable to reduce MTT. Methicillin-resistant Staphylococcus aureus exposed to flavonoids from Marchantia convoluta showed a flavonoids concentration-dependent inhibition of the ability to reduce MTT. In the assay, less than 12 h was required to attain susceptibility results and fewer bacteria were utilized than in traditional methods. The RLS technique could, in combination with the MTT assay, be a rapid and sensitive measuring method to determine the in vitro activity of new antimicrobials.

A Continuous Spectrophotometric Assay for NADPH-cytochrome P450 Reductase Activity Using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide

  • Yim, Sung-Kun;Yun, Chul-Ho;Ahn, Tae-Ho;Jung, Heung-Chae;Pan, Jae-Gu
    • BMB Reports
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    • v.38 no.3
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    • pp.366-369
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    • 2005
  • NADPH-cytochrome P450 reductase (CPR) transfers electrons from NADPH to cytochrome P450 and also catalyzes the one-electron reduction of many drugs and foreign compounds. Various spectrophotometric assays have been performed to examine electron-accepting properties of CPR and its ability to reduce cytochrome $b_5$, cytochrome c, and ferricyanide. In this report, reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) by CPR has been assessed as a method for monitoring CPR activity. The principle advantage of this substance is that the reduction of MTT can be assayed directly in the reaction medium by a continuous spectrophotometric method. The electrons released from NADPH by CPR were transferred to MTT. MTT reduction activity was then assessed spectrophotometrically by measuring the increase of $A_{610}$. MTT reduction followed classical Michaelis-Menten kinetics ($K_m\;=\;20\;{\mu}M$, $k_{cat}\;=\;1,910\;min^{-1}$). This method offers the advantages of a commercially available substrate and short analysis time by a simple measurement of enzymatic activity of CPR.