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 cm2 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 R2 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.
Cell Count ; Cell Line ; Humans ; Radiation Tolerance* ; Tetrazolium Salts

OBJECTIVE: To compare inhibition of cell growth and apoptosis in human cervical cancer cell lines (CaSki) by paclitaxel, cisplatin, arsenic trioxide and tetraarsenic oxide. METHODS: Inhibition of cell growth was determined by the water-soluble tetrazolium salts (WSTs) -1 assay. For apoptosis analysis in CaSki cell line treated with single or combination of two agents, CaSki cell line treated with each agent was stained with annexin-V/PI and flow cytometry was performed. RESULTS: Progression of apoptosis in CaSki cell line treated with paclitaxel, cisplatin, arsenic trioxide, and tetraarsenic oxide was time dependent. Inhibition of cell growth in CaSki cell line by paclitaxel, cisplatin, arsenic trioxide, and tetraarsenic oxide was dose and time dependent. Especially, tetraarsenic oxide was more effective in inhibition of CaSki cell growth compared to arsenic trioxide. Group treated with combination of cisplatin and tetraarsenic oxide showed more progressive apoptosis compared to other combination group. CONCLUSION: Tetraarsenic oxide has more potent anti-tumor effects than other agents on CaSki cell line. We need to consider further study about antitumor effect of tetraarsenic oxide through clinical study.
Apoptosis ; Arsenic ; Arsenicals ; Cell Line ; Cisplatin ; Flow Cytometry ; Humans ; Oxides ; Paclitaxel ; Tetrazolium Salts ; Uterine Cervical Neoplasms

In gastric adenocarcinoma, high rates of loco-regional recurrences have been reported even after complete resection, and various studies have been tried to find the role of postoperative adjuvant therapy. Among them, Intergroup 0116 trial was a landmark trial, and demonstrated the definite survival benefit in adjuvant chemoradiotherapy, compared with surgery alone. However, the INT 0116 trial had major limitation for global acceptance of the INT 0116 regimen as an adjuvant treatment modality because of the limited lymph node dissection. Lately, several randomized studies that were performed to patients with D2-dissected gastric cancer were published. This review summarizes the data about patterns of failure after surgical resection and the earlier prospective studies, including INT 0116 study. Author will introduce the latest studies, including ARTIST trial and discuss whether external beam radiotherapy should be applied to patients receiving extended lymph node dissection and adjuvant chemotherapy.
Adenocarcinoma ; Chemoradiotherapy, Adjuvant ; Chemotherapy, Adjuvant ; Humans ; Lymph Node Excision ; Radiotherapy, Adjuvant ; Recurrence ; Stomach Neoplasms ; Tetrazolium Salts

In gastric adenocarcinoma, high rates of loco-regional recurrences have been reported even after complete resection, and various studies have been tried to find the role of postoperative adjuvant therapy. Among them, Intergroup 0116 trial was a landmark trial, and demonstrated the definite survival benefit in adjuvant chemoradiotherapy, compared with surgery alone. However, the INT 0116 trial had major limitation for global acceptance of the INT 0116 regimen as an adjuvant treatment modality because of the limited lymph node dissection. Lately, several randomized studies that were performed to patients with D2-dissected gastric cancer were published. This review summarizes the data about patterns of failure after surgical resection and the earlier prospective studies, including INT 0116 study. Author will introduce the latest studies, including ARTIST trial and discuss whether external beam radiotherapy should be applied to patients receiving extended lymph node dissection and adjuvant chemotherapy.
Adenocarcinoma ; Chemoradiotherapy, Adjuvant ; Chemotherapy, Adjuvant ; Humans ; Lymph Node Excision ; Radiotherapy, Adjuvant ; Recurrence ; Stomach Neoplasms ; Tetrazolium Salts

BACKGROUND: This experiment was performed to determine the effect of polyphenolic (-)-epigallocatechin (EGCG), the most abundant catechin of green tea, given at reperfusion period. METHODS: Isolated rat hearts were subjected to 30 min of regional ischemia and 2 h of reperfusion. Green tea extract (GT) was perfused with the following concentrations; 0, 0.5, and 1 micrometer (GT-O, GT-0.5, and GT-1, respectively). In a next experiment, hearts were assigned randomly to one of the following groups; Control, EGCG-1 (1 micrometer of EGCG), and EGCG-10 (10 micrometer of EGCG). GT and EGCG were perfused for a period of 5 min before and 30 min after reperfusion. For comparison of cardioprotection among groups, morphometric measurement was performed by 2,3,5-triphenyltetrazolium chloride staning. RESULTS: GT 1 micrometer (10.3 +/- 2.1%, P < 0.05) significantly reduced infarct volume as a percentage of ischemic volume compared to untreated hearts (27.4 +/- 1.1%). EGCG 10 micrometer (13.2 +/- 4.0%) significantly reduced myocardial infarction compared to control hearts (27.2 +/- 1.4%, P = 0.002). After 2 h of reperfusion, cardiodynamic variables, including left ventricular developed pressure, rate-pressure produce, +dP/dt(max), and -dP/dt(min) were significantly improved by 10 micrometer of EGCG compared to control hearts (P = 0.01, 0.016, 0.009, and 0.019, respectively). CONCLUSIONS: EGCG treatment at an early reperfusion period reduces myocardial infarction and improves cardiodynamics in isolated rat hearts.
Animals ; Catechin ; Heart ; Ischemia ; Myocardial Infarction ; Myocardial Reperfusion ; Myocardium ; Rats ; Reperfusion ; Reperfusion Injury ; Tea ; Tetrazolium Salts

BACKGROUND: This experiment was performed to determine the effect of polyphenolic (-)-epigallocatechin (EGCG), the most abundant catechin of green tea, given at reperfusion period. METHODS: Isolated rat hearts were subjected to 30 min of regional ischemia and 2 h of reperfusion. Green tea extract (GT) was perfused with the following concentrations; 0, 0.5, and 1 micrometer (GT-O, GT-0.5, and GT-1, respectively). In a next experiment, hearts were assigned randomly to one of the following groups; Control, EGCG-1 (1 micrometer of EGCG), and EGCG-10 (10 micrometer of EGCG). GT and EGCG were perfused for a period of 5 min before and 30 min after reperfusion. For comparison of cardioprotection among groups, morphometric measurement was performed by 2,3,5-triphenyltetrazolium chloride staning. RESULTS: GT 1 micrometer (10.3 +/- 2.1%, P < 0.05) significantly reduced infarct volume as a percentage of ischemic volume compared to untreated hearts (27.4 +/- 1.1%). EGCG 10 micrometer (13.2 +/- 4.0%) significantly reduced myocardial infarction compared to control hearts (27.2 +/- 1.4%, P = 0.002). After 2 h of reperfusion, cardiodynamic variables, including left ventricular developed pressure, rate-pressure produce, +dP/dt(max), and -dP/dt(min) were significantly improved by 10 micrometer of EGCG compared to control hearts (P = 0.01, 0.016, 0.009, and 0.019, respectively). CONCLUSIONS: EGCG treatment at an early reperfusion period reduces myocardial infarction and improves cardiodynamics in isolated rat hearts.
Animals ; Catechin ; Heart ; Ischemia ; Myocardial Infarction ; Myocardial Reperfusion ; Myocardium ; Rats ; Reperfusion ; Reperfusion Injury ; Tea ; Tetrazolium Salts

MTT assay is commonly used to assess the cellular cytotoxicity caused by anticancer drugs in glioblastomas. However, there have been some reports insisting that MTT assay exhibited non-specific intracellular reduction of tetrazolium which led to underestimated results of cytotoxicity. Here, we examine whether or not MTT assay can lead to incorrect information regarding alcohol-induced cytotoxicity on immortalized and primary glioblastoma cells. MTT assay was applied to assess the ethanol-induced cytotoxicity at various ethanol concentrations. The cellular cytotoxicity induced by different doses of ethanol was analyzed and compared through several cytotoxic assays. Ethanol-induced cytotoxicity observed through MTT assay on both cell types was shown to be ethanol dose-dependent below a 3% concentration. However, the cytotoxicity was shown to be markedly underestimated only in primary cells at a 5% concentration. RT-PCR and Western Blot showed increased expressions of pro-apoptotic proteins and decreased expressions of anti-apoptotic proteins in an ethanol dose-dependent manner in both cell types. Furthermore, we present a possible mechanism for the unreliable result of MTT assay. A high concentration of ethanol induces more severe membrane damage and increased intracellular concentration of NADH in primary cells which enhances the nonspecific reduction of tetrazolium salt. Together, our findings demonstrate that the cytotoxicity on primary cells could inaccurately be assessed when detected through MTT assay. Therefore, a careful interpretation is needed when one would analyze the cytotoxic results of MTT assay, and it is suggested that other assays must be accompanied to produce more reliable and accurate cytotoxic results on primary glioblastoma cells.
Apoptosis Regulatory Proteins ; Blotting, Western ; Ethanol ; Glioblastoma* ; Membranes ; NAD ; Tetrazolium Salts

BACKGROUNDThe cytotoxicity of dermal substitutes may be increased by the very processes used to deplete the cells. The present research aimed to investigate the method for monitoring the cytotoxicity of cell-free dermal substitutes using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method.

METHODSThe cytotoxicity of four dermal substitutes was evaluated using the MTT method according to the standards set by the Chinese State Food and Drug Administration (SFDA). Swine acellular dermal matrix (SADM) and goat acellular dermal matrix (GADM) were produced using a repeated freeze-thaw method. Human dermal matrix glutaraldehyde composite (HADM-G) and SADM cross-linked with glutaraldehyde (SADM-G) were produced using conventional methods.

RESULTSThe cytotoxicity of all dermal substitutes ranged from Grade 0 to Grade 1, meeting the standards of the Chinese FDA. The OD(490) of both SADM and GADM was higher than that of either HADM-G or SADM-G (P < 0.05).

CONCLUSIONDermal substitutes produced by the freeze-thaw method are less cytotoxic than those produced using conventional methods.

It is known that ischemic cerebrovascular disease is causing enormous harm to human health on account of the resultant high morbidity and disability rate. In this connexion, the anticipated target is to control the size of focal ischemic cerebral infarction, which is also an important method for judgment of therapeutic efficacy. The key question is to survey the size accurately and objectively; at the same time, the quantitative analysis of focal ischemic cerebral infarction is the pivotal question affecting the experiment conclusion and the reliability level. In this paper are introduced and summarized the methods being recently and commonly used in survey and computation, and the studies made on quantitative analysis of focal ischemic cerebral infarction by 2, 3, 5-triphenyltetrazolium chloride (TTC) staining method. Also are summarized the principles of dyeing in TTC method, the preparatory work, and the commonly used method of surveying and computation. It is the intent of this review to provide relevant data and suggestion for research workers.
Animals ; Brain Ischemia ; pathology ; Cerebral Infarction ; pathology ; Coloring Agents ; Humans ; Reperfusion Injury ; pathology ; Tetrazolium Salts

OBJECTIVETo investigate the effects of bioactive glasses (BG) including 45S5 and nano-58S on proliferation, angiogenic markers vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) secretion and gene expression of human dental pulp cells (HDPC).

METHODSHDPC of 4th passage were cultured in Dulbecco's modified Eagle's medium (DMEM) which contained 0.1 g/L 45S5 or nano-58S ionic dissolution products. Meanwhile HDPC were cultured in DMEM without BG as control group. Proliferation of the cells was evaluated with methyl thiazolyl tetrazolium (MTT) assay on day 1, 2, 3. Quantitative real-time PCR and quantitative sandwich enzyme immunoassays were used to test VEGF and bFGF gene expression and protein secretion of HDPC on day 1, 2, 3.

RESULTSThe relative growth rate (RGR) of 45S5 and nano-58S groups were (134.5 ± 5.0)% and (146.3 ± 19.8)%, which was significantly different from that of control group (P < 0.05). The quantity of VEGF secretion of two experimental groups were (189.29 ± 4.64) and (216.18 ± 14.67) ng/L, respectively, significantly higher than that of the control group [(159.03 ± 11.69) ng/L] (P < 0.05). Furthermore, the nano-58S group secreted much more VEGF than 45S5 group (P < 0.05).bFGF secretion of HDPC was also enhanced by both 45S5 and nano-58S bioactive glasses. The VEGF gene expression of 45S5 and nano-58S on day 1 were (1.70 ± 0.19) and (1.63 ± 0.42), while the bFGF gene expressin on day 3 were (1.49 ± 0.02) and (2.30 ± 0.04), all significantly higher than that of control group (P < 0.05).

CONCLUSIONSBioactive glasses can enhance the proliferation, VEGF and bFGF secretion and gene expression of human dental pulp cells. Compared with 45S5, nano-58S showed a higher activation.