PURPOSE: To recognize the anatomical positions of the superior oblique muscle in enucleated eyes using trypan blue. METHODS: Twenty-two surgically-enucleated eyes of 11 bodies were studied. The shortest distance from the nasal insertion of superior rectus to the anterior end of the superior oblique tendon, the distance from the temporal insertion of superior rectus to the anterior end of the superior oblique insertion, and the greatest width of superior oblique tendon insertion were measured by caliper 3 consecutive times. The average values in each of the above 3 points were calculated, and values prior to and after trypan blue staining were compared. RESULTS: Prior to staining with trypan blue, the average distance from the nasal insertion of superior rectus to the anterior end of the superior oblique tendon was 4.97 mm and the average distance from the temporal insertion of superior rectus to the anterior end of the superior oblique insertion was 7.57 mm; after staining with trypan blue, the average values were 5.09 mm and 7.65 mm, respectively. There was no statistically meaningful difference in values prior to and after staining (p > 0.05). Prior to staining, the average value of the greatest width of the superior oblique tendon was 10.32 mm, and after staining with trypan blue, the average value increased to 10.76 mm. There was a statistically meaningful difference between the values (p = 0.02). CONCLUSIONS: Trypan blue staining helped to recognize the location and the width of the superior oblique tendon more precisely.
Diminazene ; Eye ; Muscles ; Tendons ; Trypan Blue

The effects of topical sodium-hyaluronan on the stromal and endothelial healing were examined in the repair process of experimental corneal alkali wounds. Corneal alkali wounds were produced in one eye of each rabbit by applying a 5.5 mm round filter paper soaked in 1N NaOH onto the central cornea for 60 seconds. Then the eyes were treated topically with either 1% Na-HA(the treatment group)or a phosphate buffered saline(PBS)(the con-trol group)4 times per day for 3 weeks. Endothelial wound morphometry was performed after alizarin red and trypan blue staining. The stromal healing was assessed by counting polymorphonuclear leukocytes(PMNs)and keratocytes in the central and marginal wounds areas. The stroma treated with Na-HA had less PMNs than that of the control group during the early healing period. The defect area of the endothelium was significantly smaller in the Na-HA treated group than in the control corneas. The present findings indicate that topically applied 1%Na-HA affects stromal and endothelial healing during the early repair process after corneal alkali wounds.
Alkalies* ; Cornea ; Endothelium ; Trypan Blue ; Wounds and Injuries*

PURPOSE: To evaluate the effects of trypan blue (TB) on the survival of cultured human trabecular meshwork cells (HTMCs). METHODS: Primarily cultured HTMCs were exposed to 0.05, 0.10 or 0.50% TB for 1, 5 or 30 min. Cellular survival was assessed using the MTT assay and degree of apoptosis was analyzed with flow cytometry using annexin-V/propidium iodide double staining. RESULTS: Long-term exposure or high concentration of TB decreased the survival of HTMCs (p < 0.05). In flow cytometric analysis, exposure to 0.50% TB for 30 min increased the degree of apoptosis (p < 0.05). Commercial TB decreased cell survival after exposure for 5 min and increased the degree of apoptosis after exposure for 30 min (p < 0.05). CONCLUSIONS: TB may cause cellular damage of cultured HTMCs and apoptosis could be the underlying mechanism. In TB-assisted cataract surgery, TB should be used for the shortest time possible and removed completely.
Apoptosis ; Cataract ; Cell Survival ; Flow Cytometry ; Humans ; Trabecular Meshwork* ; Trypan Blue*

The effects of topical dexamethasone on the endothelial healing and the change of aqueous composition were examined in the repair process of experimental corneal alkali wounds. Corneal alkali wounds were induced, then the eyes were treated topically with either 0.1%dexamethasone or abalanced salt solution[BSS]4 times per day for 8 weeks. Endothelial wound morphometry was performed after alizarin red and trypan blue staining. The concentrations of ascorbic acid, glucose, and the ions, Na, K , Ca2 and Mg2 , were measured in the aqueous humor. Endothelial healing in control corneas showed a biphasic pattern of healing:an initial short-term healing for the first week and then a late long-term healing following a secondary endothelial breakdown. Topical administration of 0.1%dexamethasone deterred endothelial healing during the early period and prevented secondary endothelial breakdown. Total repair process of endothelium was accelerated by the dexamethasone treatment. Among the various components of the aqueous humor examined, ascorbic acid seemed most sensitive to change caused by the alkali injury and dexametha-sone treatment. The present data indicate that dexamethasone may have a therapeutic potential in the management of endothelial healing after corneal alkali injury.
Administration, Topical ; Alkalies* ; Aqueous Humor ; Ascorbic Acid ; Cornea ; Dexamethasone ; Endothelium ; Glucose ; Ions ; Trypan Blue ; Wounds and Injuries*

PURPOSE: To explore the effect of recombinant human EGF on the proliferation and survival of human fibroblast cell lines following irradiation. MATERIALS AND METHODS: Fibroblast was originated human skin and primary cultured. The trypan blue stain assay and MTT assay were used to study the proliferative effects of EGF on human fibroblast cell lines in vitro. An incubation of fibroblasts with rhEGF for 24 hours immediately after irradiation was counted everyday. Cell cycle distributions were analyzed by FACS analysis. RESULTS: Number of fibroblast was significantly more increased rhEGF (1.0 nM, 10 nM, 100 nM, 1,000 nM) treated cell than control after 8 Gy irradiation. Most effective dose of rhEGF was at 160 nM. These survival differences were maintained at 1 week later. Proportion of S phase was significantly increased on rhEGF treated cells. CONCLUSION: rhEGF cause increased fibroblast proliferation following irradiation. We expect that rhEGF was effective for radiation induced wound healing.
Cell Cycle ; Cell Line* ; Epidermal Growth Factor* ; Fibroblasts* ; Humans* ; S Phase ; Skin ; Trypan Blue ; Wound Healing

BACKGROUND: Human telomerase is a ribonucleoprotein polymerase, which synthesizes telomeric repeat sequences, and human telomerase reverse transcriptase (hTERT) has been identified as the catalytic subunit, as well as the rate-limiting component, of telomerase. In this study, we attempted to identify the modulators of telomerase, and to determine the molecular mechanisms underlying cisplatin-induced apoptosis. METHODS: To determine the role of telomerase in cisplatin-induced apoptosis, we measured telomerase activity and analyzed apoptosis using PI and trypan blue staining. Also, we inhibited the caspase activations using Z-VAD-fmk to analyze the effects on expression of hTERT protein. Finally, we induced the transient co-expression of the Bcl-2 and Bak genes in HEK293 cells, and then, the telomerase activity and expression of hTERT were evaluated. RESULTS: In the Bcl-2-overexpressing HeLa cells, telomerase activity was more enhanced, and cell death was reduced to 40-50% that of the mock controls. This finding suggests that Bcl-2-induced telomerase activity exerts an antiapoptotic effect in cisplatin-induced death. As caspase activation was inhibited via Z-VAD-fmk, the hTERT protein was recovered in the mock controls, but not in the Bcl-2-overexpressing cells. This suggests that the expression of hTERT can be regulated by caspases, but Bcl-2 was located within the upstream pathway. Moreover, when the Bcl-2 and Bak genes were co-transfected into the HEK293, both telomerase activity and hTERT protein were prominently reduced. CONCLUSIONS: Bcl-2-induced telomerase activity inhibits cisplatin-induced apoptosis in HeLa cells, and can be regulated via both caspases and the interaction of Bcl-2 and Bak.
Apoptosis ; Caspases* ; Catalytic Domain ; Cell Death* ; Cisplatin ; HEK293 Cells ; HeLa Cells ; Humans* ; Ribonucleoproteins ; Telomerase* ; Trypan Blue

Human fibroblasts that maintain the structural integrity of connective tissues by secreting precursors of the extracellular matrix are typically cultured with serum. However, there are potential disadvantages of the use of serum including unnatural interactions between the cells and the potential for exposure to animal pathogens. To prevent the possible influence of serum on fibroblast cultures, we devised a serum-free growth method and present in vitro data that demonstrate its suitability for growing porcine fetal fibroblasts. These cells were grown under four different culture conditions: no serum (negative control), 10% fetal bovine serum (FBS, positive control), 10% knockout serum replacement (KSR) and 20% KSR in the medium. The proliferation rates and viabilities of the cells were investigated by counting the number of cells and trypan blue staining, respectively. The 10% FBS group showed the largest increase in the total number of cells (1.09 x 10(5) cells/ml). In terms of the rate of viable cells, the results from the KSR supplementation groups (20% KSR: 64.7%; 10% KSR: 80.6%) were similar to those from the 10% FBS group (68.5%). Moreover, supplementation with either 10% (3.0 x 10(4) cells/ml) or 20% KSR (4.8 x 10(4) cells/ml) produced similar cell growth rates. In conclusion, although KSR supplementation produces a lower cell proliferation rate than FBS, this growth condition is more effective for obtaining an appropriate number of viable porcine fetal fibroblasts in culture. Using KSR in fibroblast culture medium is thus a viable alternative to FBS.
Animals ; Cell Proliferation ; Connective Tissue ; Diminazene ; Extracellular Matrix ; Fibroblasts ; Humans ; Trypan Blue

BACKGROUND: The quality of cord blood largely depends on cell viability. Viability assessments using trypan blue or 7-aminoactinomycin (7-AAD) staining, which are commonly used methods, may not reflect early apoptosis of cord blood cells. We aimed to investigate early apoptosis in cord blood cells following elapsed time after collection using double staining with annexin V and 7-AAD and to compare the result with that of viability evaluation using trypan blue or 7-AAD staining. METHODS: Umbilical cord blood samples were obtained from 30 pregnant women at the time of delivery between July 2012 and March 2013. Viability of cord blood cells was determined at 0 (T0), 24, and 48 hr after collection by using trypan blue exclusion assay, 7-AAD staining, and 7-AAD/annexin V staining. RESULTS: Viabilities defined by 7-AAD/annexin V staining at T0, 24, and 48 hr after collection were respectively as follows: total nucleated cells, 92.8+/-4.5%, 78.4+/-7.8%, and 65.5+/-8.1%; mononuclear cells, 94.4+/-1.7%, 90.8+/-4.2%, and 84.2+/-6.7%; and CD34-positive cells, 92.4+/-3.0%, 90.7+/-4.7%, and 89.3+/-7.0%. The viability using trypan blue was more than 90% until 48 hr after collection. CONCLUSIONS: The mean viability of total nucleated cells using 7-AAD/annexin V staining decreased to less than 80% at 24 hr after collection; however, the viability of CD34-positive cells was more than 85% until 48 hr. Our study's data will provide useful information for the assessing the quality of cord blood products.
Annexin A5 ; Apoptosis ; Cell Survival* ; Female ; Fetal Blood* ; Humans ; Methods ; Pregnant Women ; Trypan Blue ; Umbilical Cord*

Intracellular ions which have a major role in cellular function have been reported to affect repair of radiation damage. Recently it has been reported that ouabain sensitizes A549 tumor cells hut not CCL-120 normal cells to radiation. Ouabain inhibits the Na+-K+-pump rapidly thus it increases intracellular Na concentration. Vanadate which is distributed extensively in almost all living organisms in known to be a Na+-K+-ATPase inhibitors. This study was performed to see any change in radiosensitivity of tumor cell by vanadate and any role of Na+-K+-ATPase in radiosensitization. Experiments have been carried out by pretreatment with vanadate in human cell line(A549, JMG) and mouse cell line(L1210, spleen). For the cell survival MTT assay was performed for A549 and JMG cell and trypan blue dye exclusion test for L120, and spleen cells. Measurements of Na+-K+-ATPase activity in control, vanadate treated cell, radiation treated cell (9 Gy for A549 and JMG, 2 Gy for L1201, spleen), and combined 10-6 M vanadate and radiation treated cells were done. The results were summarized as follows. 1. L1210 cell was most radiosensitive, and spleen cell and JMG cell were intermediate, and A549 cell was least radiosensitive. 2. Minimum or cytotoxicity was seen with vanadate below concentration of 10-6 M. 3. In A549 cells there was a little change in radiosensitivity with treatment of vanadate. However radiation sensitization was shown in low dose level of radiation i. E. 2-Gy. In JMG cells no change in radiosensitivity was noted. Both L1210 and spleen cell had radiosensitization but change was greater in tumor cell. 4. Na+-K+-ATPase activity was inhibited significantly in tumor cell by treatment of vanadate. 5. Radiation itself inhibited Na+-K+-ATPase activity of tumor cell with high Na+- K+-ATPase concention. Increase in radiosensitivity by vanadate was closely associated with original Na+-K+-ATPase contents. From the above results vanadate had little cytotoxicity and it sensitized tumor cells to radiation. Inhibitory effect of vanadate on Na+-K+-ATPase activity might be one of the contributing factors for radiosensitization to tumor cells which has greater enzyme activity than that of normal cell. It was suggested vanadate could be used as a potential radiosensitizer for tumor cells.
Animals ; Cell Survival ; Humans ; Ions ; Mice ; Ouabain ; Radiation Tolerance* ; Spleen ; Trypan Blue ; Vanadates*

BACKGROUND: Trichomonas vaginalis is a pathogenic protozoa infecting human genitourinary tract. Metronidazole is currently the drug of choice to treat T. vaginalis infection. However, because of the side effects and the occurrence of resistant strains of metronidazole, it is needed to investigate alternatives. METHODS: The antiprotozoal effect of aquatic extract from Sophora flavescens on the growth and fine structure of T. vaginalis was examined by using trypan blue exclusion assay and electron microscopy. RESULTS: One hour after the addition of 4 mg/mL extract and half hour after the addition of 5 mg/mL showed antiprotozoal effect. One to two hours after the addition of 3 mg/mL extract, the movement of flagella and axostyle had disappeared, but death of the cells had not occurred until two hours after the addition. The fine structure of the cytoplasm was also changed half an hour to two hours after addition. The number of polyribosome decreased when that of single ribosomes in the cytoplasm increased. CONCLUSION: These results indicated that S. flavescens had the antiprotozoal effect on T. vaginalis by inhibition of cell multiplication as well as an impairment of protein synthesis.
Cell Proliferation ; Cytoplasm ; Flagella ; Humans ; Metronidazole ; Microscopy, Electron ; Polyribosomes ; Ribosomes ; Sophora* ; Trichomonas vaginalis* ; Trichomonas* ; Trypan Blue