Objective: Different cryoprotectants are used for cryopreservation of ovarian tissue in patients at risk of infertility. Ethylene glycol (EG), dimethyl sulfoxide (DMSO) and propanediol (PROH) have been chosen as the basic permeable cryoprotectants due to their decreased glass-formation characteristics compared to other cryoprotectants. In the present study, the effects of two different vitrification methods on whole mouse ovarian tissue by the use of a novel staining method (TRYPAN BLUE) has been evaluated.Methods: Ovaries of 8 day-old NMRI mice were isolated and divided among the control, vitrification 1 (Vit1) and vitrification 2 (Vit2) groups. The Vit1 solution was composed ofα-MEM+20% FBS+15% EG+15% DMSO. The Vit 2 solution was composed of α-MEM+15% FBS+20% EG+20% PROH. Vit1 and Vit2 procedures were performed at 4˚C and room temperature, respectively. Warming was performed in α-MEM+20% FBS supplemented with 1M sucrose in the Vit1 group and α-MEM+15% FBS with descending concentrations of sucrose (1, 0.5, 0.25 M) in the Vit2 group. Control and vitrified warmed ovaries were put in α-MEM supplemented by 0.4% TRYPAN BLUE for 20 min, and then stained ovaries were fixed in Bouin’s fixative, serially sectioned in paraffin wax and finally quantitatively evaluated under a light microscope.Results: The highest percentage of primordial follicles was observed in the control group.There was a significant difference between the control and Vit1 groups, and between the Vit1 and Vit2 groups (p<0.05). No significant difference was observed in primary and preantral follicles between the control and vitrification groups.Conclusion: Vitrification with EG and PROH are more suitable for preservation of follicle reserves in ovaries. TRYPAN BLUE staining is a faster and easier method for evaluation of ovarian tissue.

Background: Cell viability is an important factor in radiation therapy and thus is a method to quantify the effect of the therapy.Materials and Methods: The viability of human hepatoma (HepG2) cells exposed to radiation was evaluated by both the MTT and TRYPAN BLUE assays. The cells were seeded on 96 well-plates at a density of 1 x 104 cells/well, incubated overnight, and irradiated with 1-100 Gy.Results: The cell viability was decreased in a dose- and me- dependent manner when using the TRYPAN BLUE assay, but no significant changes in the response to dose could be detected using the MTT assay. It indicated that the MTT assay was not efficient at a cell density of 1 x 104 cells/well on 96 well-plates to determine cell viability. Subsequently, the relationship between cell viability and lower cell density (1 x 103, 3 x 103, and 5 x 103 cells/well) was investigated. A cell density of 1 x 103 was found to be the most effective when using the MTT assay. Results show that the cell density is most important when using the MTT assay in 96 well-plates to follow in radiation effects. Furthermore, the radiation-induced cell viability dependent on cell density was confirmed by using the traditional Clonogenic assay.Conclusion: Our results suggest that the MTT and TRYPAN BLUE assays are rapid methods to detect radiation-induced cell viability of HepG2 cells in about 3 days as compared with 14 days of assay time in the Clonogenic assay.To obtain accurate cell viability measures using both rapid assays, an incubation me of at least 3 days is needed a6er irradiation.

Background: Evaluation of cell viability is momentous in pharmacologic and oncological research. Cell viability evaluation determines cell sensitivity and consequently treatment outcome. Various methods are available to determine cell survival. Each of these methods evaluates different endpoints. Accordingly, determining the correlation between these methods is important. In this study, in order to determine the viability of human anaplastic thyroid cancer cell line, the sensitivity of MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] assay, TRYPAN BLUE test and clonogenic assay were compared. Methods: This experimental study was performed in the Cellular and Molecular Research Center at Guilan University of Medical Sciences, Rasht, Iran from October 2016 to March 2017. The human anaplastic thyroid cancer cell line was cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS). The cultured cells were treated with melatonin, for 24 hours. Then, the viability of the cells was evaluated by MTT assay, TRYPAN BLUE test and clonogenic assay. Furthermore, plating efficiency and surviving fraction were used in order to draw survival curve in the clonogenic assay. Results: The concentration of melatonin at IC50 point was 4. 794 0. 117 millimolar (mM) in MTT assay, 4. 375 0. 894 mM in TRYPAN BLUE test and 2. 246 0. 326 mM in clonogenic assay. Comparing the IC50 values of these test revealed that C50 values obtained from MTT assay and TRYPAN BLUE test had no significant difference (P=0. 6446), while there was a significant difference between IC50 values obtained from MTT and clonogenic assays (P=0. 0032). Moreover, the IC50 values obtained from TRYPAN BLUE test and clonogenic assay were also significantly different (P=0. 0078). The results of the regression analysis of cell viability were shown a linear, positive and significant correlation between these three methods and MTT assay and TRYPAN BLUE test showed higher correlation (r=0. 99, P<0. 001). Conclusion: Based on our results, all these methods were effective to identify cytotoxicity in human anaplastic thyroid cancer cell line, while MTT assay and TRYPAN BLUE test were more sensitive than clonogenic assay.

Background and Objectives: Evaluation of cell survival is important in cellular studies and the result is decisive for the application of the test substance. Different methods are used to evaluate cell survival and the correlation between methods is important. Here, Vero cell line was used to evaluate the effects of HSV-1 virus on cell survival. Cell survival after HSV-1 infection using sensitive colorimetric methods of 3-, 4-, 5-dimethylthiazole-2-yl,-2, 5-diphenyltetrazolium bromide (MTT), and TRYPAN BLUE on Vero cell line was examined. Materials and Methods: A qualitative study was performed on Vero cell line infected with HSV-1 virus with logarithmic dilutions10-1-5 to 10 dilutions and time intervals of 0, 4, 8, 18 and 24 hours and the s in DMEM containing 2% Fetal bovine serum (FBS). Then cell viability by MTT and TRYPAN tests was examined. The survival percentage was used to plot the survival curve of the assays. Results: Different viral dilutions were used to determine the infectious dose of 50% (TCID50) of HSV1virus with invert microscope. Acyclovir at a concentration of 50 mg/mL was compared as drug control for MTT and TRYPAN BLUE. The results of regression analysis showed cell survival. Linear, positive and significant correlation was observed between the methods that showed a correlation between MTT and TRYPAN BLUE methods (p< 0. 001, r = 0. 9( Conclusions: The results showed that both methods can be used to determine cell survival and the results of MTT can be confirmed by TRYPAN BLUE method.

Objective: The rate determination of cellular proliferation and cell viability is critical to the assessment of the effects of drugs on cells and there are different standard methods in evaluation of cell proliferation. Calprotectin is a cytosol metaloprotein in the neutrophil, macrophase, lymphocyte and monocyte cytosol that induce growth-inhibitory against various cell types including tumor cells. These finding indicate it can be as a therapeutic agtent in cancer diseases. Biological characteristics of calprotectin are contained antibacterial, antifungal, immonoregulation, chemotactic activity and inhibition of cell proliferation. In the present study, two staining assays; TRYPAN BLUE and MTT in vitro evaluation of human calprotectin proliferation inhibition on human gastric cancer cells was compared. Materials and Methods: Calprotectin was purified from human neutrophil by chromatography methods. The human gastric adenocarcinoma cell line (AGS) was used. These cells were incubated in RPMI 1640 medium supplemented with 10% FBS in a humidified incubator (37oC & 5% CO2). AGS cells (10000 cell per well) were incubated to different concentration of calprotectin (25, 50, 100, 200, 400 mg/ml) at 24, 48, 72 h. In this study TRYPAN BLUE dye exclusion and MTT assay were used for evaluation of cell proliferation inhibition.Results: Results of cytotoxicity effect of calprotectin on AGS cells show that there are significant correlation between two methods (P<0.01) and LC50 value of calprotectin calculated by MTT assay is lower than TRYPAN BLUE in all time intervals. The LC50 of calprotectin for AGS cell at 24, 48 and 72 h was determined 33.29, 71 and 141.8 mg/ml by TRYPAN BLUE and 96.78, 38.66 and 9.86 mg/ml by MTT assay, respectively. There are significant correlation between both methods at different time intervals and it is positive linear (P<0.01–P<0.001). Conclusion: Result of cell cyto toxicity of calprotectin by both methods indicated that correlation is positive and significant. It can be said that MTT assay is more sensitive, easy to handle, a large number of probes can be assayed in a relatively short time.

Breast cancer is the most common cancer in the Iranian women. Essential oil of thymus is containing anticancer compounds including thymol. The aim if this research was studding the effect of thymus essential oil on the breast cancer cell line MCF-7 using MTT and TRYPAN BLUE methods, and comparing of its effect with taxol as a well-known anticancer agent. In this research, MCF-7 and Hu-02 cell lines were porched from IBRC institute. The cells were treated by different concentrations of thymus essential oil and taxol in the times of 24, 48 and 72 hours. The cytotoxicity of the treatments was measured by means of MTT and TRYPAN BLUE staining. Results showed that Thymus essential oil had cytotoxic effect, in different doses, on the MCF-7 cell line. The highest cytotoxic effect was measured in the group treated by 125mg/ml of thymus essential oil. The Thymus essential did not show any cytotoxic effect on the cells of HU-02. Results were also showed that the essential oil can increase the cytotoxicity effect of Taxol on the cancer cell line, thus it seems that is effective in breast cancer therapy; probably, it causes to cell death by apoptosis induction.

Nanoparticles of perovskite-type LaCo0.5Fe0.5O3 (LCFO) were fabricated by sol–gel method. Thermal decomposition process of the complex precursor was examined by means of differential thermal analysis–thermal gravimetric analysis (DTA/TGA). X-ray diffraction (XRD) results showed that single perovskite phase has been completely formed after calcination at 750°C. In addition, the surface morphology and composition of these nano powders were also investigated using SEM and EDX. These nanoparticles showed the excellent adsorption efficiency towards TRYPAN BLUE (TB) as a reactive dye in aqueous solution. The adsorption studies were carried out at different pH values, dye concentrations, various adsorbent dosages and contact time in a batch experiments. LCFO exhibited good dye removal efficiency at acidic pH specially pH 2. Experimental results indicated that the adsorption kinetic data follow a pseudo-second-order rate for tested dye. The isotherm evaluations revealed that the Freundlich model better fits to the experimental equilibrium data than Langmuir the model.

Background and purpose: Breast cancer is the most common cancer in woman. About one million new cases are diagnosed every year worldwide. Many researchers are interested in anti-cancer effects of natural products. In this research, cytotoxic effects of Crocus caspius extract against breast cancer and normal cell lines were investigated and total phenols and flavonoids were also determined.Materials and methods: Flowers of Crocus caspius were collected from Neka in Mazandaran province, Iran, in autumn 2014. The powder of dried flowers were macerated with 80% methanol, three times every 48 hr. The methanol extract was concentrated under reduced pressure. Cell viability was determined using MTT and TRYPAN BLUE assay was done on breast carcinoma cell lines (MCF7, 4T1, and SKBR3) and Swiss mouse embryo fibroblast (NIH/3T3) as normal cell lines. Various concentrations (1, 50, 100, 500, and 1000 mg/ml) of the extract and positive control were examined for determination of IC50.Results: Crocus caspius extract showed no considerable cytotoxic effects against breast cancer and normal cell lines. Total phenolic and flavonoid contents of the extract were quite high; 238.25±4.35 mg GAE/g and 85.41±6.24 mg quercetin/g of dry extract, respectively. Conclusion: These results indicated non toxicity of Crocus caspius extract. Further studies are needed to investigate the preventive effect of C.caspius extract against cancer according to its total phenolic and flavonoid contents.

Background: There is a growing interest in understanding the biological effects of timetested folk medicinal plants including the green leafy vegetables, which supply minerals and vitamins to the diet. Trigonella foenum-graecum L (fenugreek) is a dietary vegetable and there are reports concerning its antinociceptive effects in Iranian traditional medicine. Its seeds are also known for their carminative, tonic, antidiabetic, antineoplastic and restorative properties. These reports and the hypoglycemic effect of fenugreek leaf extract encouraged us to assay fenugreek aqueous extract for cytotoxicity on NIH3T3 mouse fibroblast cells.Methods: The NIH3T3 cell line was purchased from National Research Center for Genetic Engineering and Biotechnology of Iran. The cells were plated in 24-well microtiter plates with DMEM+F12 medium containing 10% fetal calf serum supplemented with 445 mg/L Lglutamine and maintained at 37°C with 5% CO2/95% air. Following a 24-hr incubation period, various concentrations (0.01-20 mg) of the extract to the culture wells. Cell viability was assessed using TRYPAN BLUE and MTT assays after five days of incubation. Results: The results show that the IC50 of the fenugreek extract as calculated from the TRYPAN BLUE and MTT assays were 1.25 and 2.5 mg/mL, respectively. Conclusions: Our findings, therefore, suggest that the aqueous extract of fenugreek is classified as nontoxic. This observed cytotoxicity is not specific and could be due to membrane disturbances.

Tumor growth is characterized by uncontrolled cell division. For centuries, silver kushta powder, composed of nano and submicro silver particles, has been used in traditional Iranian and Pakistani medicine for the treatment of melanoma and breast cancer. We have found that these nanocomposite particles are similar to silver nanoparticles (AgNps) in size and shape and that there are so differences in their physicochemical properties such as silver content.In the present study, a comparison of cytotoxic effects of nanosilver (AgNps) and two silver kushtas [Iranian silver kushta (IKAg) and Pakistan silver kushta (PKAg)] have been conductedin different concentrations against tumor cell lines (MCF-7, HepG2, A549) and a normal cell line (MDBK) using MTT and TRYPAN BLUE exclusion tests.At first, Particle size was analyzed using the Malvern Zetasizer. The Z average diameters of samples (AgNps, PKAg, and IKAg) were64.08, 51.72 and 190.4 nm, respectively.The result of MTTtest showed no toxicity of both silver kushtas (IKAg& PKAg) toward the cancer cell lines and MDBK cells. The IC50 values of AgNpsdetermined for A549, HPG2, MCF-7, and MDBK were 5.94, 1.41, 3.68, and, 1.9 ppm, respectively. According to TRYPAN BLUE (0.2% w/v) exclusion test, the cytotoxicity of the silver materials toward primary rat hepatocytes followed the order AgNps (100%)>AgNO3 (80.9%)>Pakistani silver kushta (48.35%)>Iranian silver kushta (45%).This result illustrated that the silver components [(IKAg) & (PKAg)] of the traditional kushtas do not penetrate the cancer cell membrane and do not show cytotoxicity. Therefore, kushtas are ineffective as anti-cancer agents. However, AgNps shows good anticancer properties.