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

To obtain basic information on the detection of cellulolytic activity in Auricularia auricula-judae, the influences of dye reagent, pH, and temperature were assessed. Chromogenic dye (congo red, phenol red, remazol brilliant blue, and trypan blue) was individually incorporated into a medium containing either carboxymethyl-cellulose, Avicel, or D-cellobiose as a polysaccharide carbon substrate. The other assessments utilized pHs ranging from 4.5 to 8.0 and temperatures from 15~35degrees C. Overall, when A. auricula-judae species were transferred onto media contained Congo red and adjusted pH 7.0 and then incubated at 25degrees C for 5 days, the clear zone indicative of cellulolytic activity was more pronounced.
Benzenesulfonates ; Carbon ; Cellulose ; Congo Red ; Diminazene ; Hydrogen-Ion Concentration ; Phenolsulfonphthalein

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 viability of cord blood is an important measure of product quality. Trypan blue (TB) stain is the most commonly and conveniently used method to measure the viability of the cord blood. Recently, cytometric analysis using 7-Aminoactinomycin D (7-AAD) was introduced. Staining with 7-AAD is more sensitive in detecting cellular damage than staining with TB. In addition to this, 7-AAD allows specific measurement of the viability of total nucleated cells (TNC), mononuclear cells (MNC) and CD34+ cells. In this study, we compared the viability of TNC between the TB and 7-AAD method, as well as analyzing the viability of each cell population. METHODS: From February to July 2010, 102 cord blood units were collected and assessed for the viability of TNC by the TB and 7-AAD methods. The viability of mononuclear cells (MNC) and CD34+ cells was assessed by 7-AAD method. RESULTS: The TB and 7-AAD methods were used to assess the viability of TNC, which was 90.1+/-5.7% and 68.4+/-8.0%, respectively. The viability of MNC and CD34+ cells measured by the 7-AAD method was 91.8+/-4.3% and 93.4+/-5.1%, respectively. CONCLUSION: The TNC viability of 7-AAD method was significantly lower than that of TB method. In 7-AAD method, the viabilities of MNC and CD34+ cells were significantly higher than that of TNC. As those are important prognostic factors and measures for successful engraftment after the transplantation, the measurement of the viabilities of MNC and CD34+ cells by 7-AAD method would be helpful to the quality control of the cord blood product.
Cell Survival ; Cryopreservation ; Dactinomycin ; Diminazene ; Fetal Blood ; Quality Control ; Transplants ; Trypan Blue ; Umbilical Cord

BACKGROUND: On performing umbilical cord blood (UCB) transplantation, faster engraftment may lead better clinical outcome. Because transplanted viable cell count in UCB is related to the engraftment, accurate evaluation of viability of CD34+cells in cryopreserved UCB has clinical implication. We examined the difference in viability of cells in cryopreserved UCB according to the duration of cryopreservation and different methods. METHODS: A total of 60 UCB samples which were cryopreserved for 1 to 4 years were used in this study. Viability of cryopreserved cells were examined with trypan blue exclusion assay, DNA contents analysis, caspase-3 activation test, intracellular esterase activity and Annexin-V/PI staining. RESULTS: After thawing the cryopreserved UCB, 89% of the total MNCs and 84% of CD34+cells were viable as identified by trypan blue exclusion assay. In the CD34+cell population, the cell death rate was found to be 47% by Annexin-V/PI staining and less than 5% by DNA contents analysis. However, cspase-3 activity failed to document apoptosis. The intracellular esterase activity test also showed a cell death rate of about 10~20% at 2, 4, and 6 hours after thawing. CONCLUSION: Viable cells in UCB should be measured by several compensatory techniques rather than a single method. Discordance among Annexin-V/PI staining versus trypan blue exclusion, DNA contents analysis, and the caspase-3 activation test or intracellular esterase activity should be clarified in order to apply these techniques for actual cord blood transplantation.
Apoptosis ; Caspase 3 ; Cell Count ; Cell Death ; Cryopreservation ; Diminazene ; DNA ; Fetal Blood ; Transplants ; Trypan Blue

To determine the optimal media conditions for the detection of the extracellular cellulase activity in Ganoderma neo-japonicum, we varied three media conditions: dye reagent, pH, and temperature. We evaluated the use of four dyes, Congo red, phenol red, remazol brilliant blue, and trypan blue. To observe the effect of pH on the chromogenic reaction, we tested media ranging from 4.5 to 8.0. To research the effect of temperature on the clear zone and the fungus growing zone, we tested temperatures ranging from 15 to 35degrees C. On the whole, the best protocol called for Ganoderma neo-japonicum transfer onto media containing Congo red with a pH of 7.0, followed by incubation at 25degrees C for 5 days. Our results will be useful to researchers who study extracellular enzyme activity in Ganoderma neo-japonicum.
Benzenesulfonates ; Cellulase ; Coloring Agents ; Congo Red ; Diminazene ; Fungi ; Ganoderma ; Hydrogen-Ion Concentration ; Phenolsulfonphthalein ; Trypan Blue

To determine the optimal medium conditions for the detection of the cellulolytic activity in Ganoderma lucidum, we varied three media conditions: dye reagent, pH, and temperature. First, we evaluated the use of four dyes, Congo Red, Phenol Red, Remazol Brilliant Blue, and Trypan Blue. To observe the effect of pH on the chromogenic reaction, we also made and tested various media spanning acidic and alkaline pHs, ranging from 4.5 to 8.0. Furthermore, in order to research the effect of temperature on the clear zone and the fungus growing zone, we tested temperatures ranging from 15 to 35degrees C. On the whole, the best protocol called for Ganoderma lucidum transfer onto media containing Congo red with pH adjusted to 7.0, followed by incubation at 25degrees C for 5 days. Our results will be useful to researchers who aim to study extracellular enzyme activity in Ganoderma lucidum.
Benzenesulfonates ; Coloring Agents ; Congo Red ; Diminazene ; Fungi ; Ganoderma ; Hydrogen-Ion Concentration ; Phenolsulfonphthalein ; Reishi ; Trypan Blue

Mycobacterial cell-wall skeleton (CWS) is an immunoactive and biodegradable particulate adjuvant and has been tried to use for immunotherapy. The CWS of Mycobacterium bovis bacillus Calmette-Guerin (BCG-CWS) was studied as an universal vaccine vehicle for antigen conjugation, to develop potentially effective and safe vaccine. Although a variety of biological activities of BCG-CWS have been studied, the effects of BCG-CWS on spleen cells are not fully elucidated. Using MTT assay and trypan blue exclusion test, we found that BCG-CWS significantly enhanced the viability and proliferation of cells. Multiple clusters, indicating proliferation, were observed in BCG-CWS-treated spleen cells and surface marker staining assay revealed that BCG-CWS promoted the proliferation of CD19+ B lymphocyte rather than CD4+ or CD8+ T lymphocyte. In addition, BCG-CWS up-regulated the expression of anti-apoptotic molecules such as bcl-2, bcl-xL. BCG-CWS increased the surface expression of CD25 and CD69 as well as IL-2 production of spleen cells, suggesting increased activation. Furthermore, BCG-CWS enhanced the antigen-specific cell proliferation and interferon-gamma production of spleen cells. Taken together, these results demonstrate the immunostimulatory effects of BCG-CWS on spleen cells via multiple mechanisms, providing valuable information to broaden the use of BCG-CWS in clinical and research settings.
Animals ; Bacillus ; Cell Proliferation ; Collodion ; Diminazene ; Immunotherapy ; Interferon-gamma ; Interleukin-2 ; Lymphocytes ; Mice ; Mycobacterium bovis ; Skeleton ; Spleen ; Trypan Blue

A 2-year old castrated male Alaskan malamute was referred with primary complaints of marked anemia, hemeglobinuria and depression. Laboratory tests revealed canine babesiois with severe anemia. The dog was treated by blood transfusion and beneril (diminazene aceturate, 3.5 mg/kg IM). Two days after Beneril injection, the dog suddenly showed ataxia progressing to paresis. MRI revealed irregularly diffused lesions in the cerebellum. The case was tentatively diagnosed as cerebellar encephalopathy caused by diminazene toxicity. The dog successfully recovered following steroid therapy.
Anemia ; Animals ; Ataxia ; Blood Transfusion ; Cerebellar Ataxia ; Cerebellum ; Depression ; Diminazene* ; Dogs* ; Humans ; Magnetic Resonance Imaging ; Male ; Paresis

A 2-year old castrated male Alaskan malamute was referred with primary complaints of marked anemia, hemeglobinuria and depression. Laboratory tests revealed canine babesiois with severe anemia. The dog was treated by blood transfusion and beneril (diminazene aceturate, 3.5 mg/kg IM). Two days after Beneril injection, the dog suddenly showed ataxia progressing to paresis. MRI revealed irregularly diffused lesions in the cerebellum. The case was tentatively diagnosed as cerebellar encephalopathy caused by diminazene toxicity. The dog successfully recovered following steroid therapy.
Anemia ; Animals ; Ataxia ; Blood Transfusion ; Cerebellar Ataxia ; Cerebellum ; Depression ; Diminazene* ; Dogs* ; Humans ; Magnetic Resonance Imaging ; Male ; Paresis