“Dye another day!” – dyes in ophthalmology

Background: Dyes are substances that are an integral part of ocular procedures and surgeries. In Clinical practice, dyes help in better visualization and aid in diagnoses of ocular surface disorders. In Surgical practice, dyes help in better resolution of the structures that are otherwise naked to the surgeon’s eyes. Purpose: To educate ophthalmologists about the importance and uses of dyes. Synopsis: Dyes have become an important part of an ophthalmologists' clinical as well as surgical practice. This video aims at educating the different characteristics, uses, advantages and disadvantages of each dye. Dyes help in identifying the obscure and highlighting the invisible. The indications and contraindications as well as the side effects of each dye are discussed which would help ophthalmologists in the correct usage of these wonder substances. This video will also help the new eye doctors understand and utilize these dyes judiciously which would aid in their learning process and provide better patient care. Highlights: This video highlights the uses, indications, contraindications and side effects of all the dyes used in ophthalmology

A Meta-analysis of the effect of trypan blue capsule staining on corneal endothelial cells in phacoemulsification / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM:To systematically evaluate the effect of trypan blue capsule staining on corneal endothelial cells in phacoemulsification. <p>METHODS: RCTs on the use of trypan blue for capsular staining in phacoemulsification were retrieved from China Knowledge Network(CNKI), Wanfang Database, Weipu Database, SinoMed, PubMed, SpringerLink, Clinicalkey, Medline, Cochrane Library, Web of Science, OVID, Embase. The search time was from the establishment of the databases to April 2019.The Meta-analysis of the included literature was made by Revman 5.3 and R 3.7.<p>RESULTS: Eight trials included 378 eyes were selected. Meta-analysis showed that the number of corneal endothelial cell loss between 0.02%, 0.06% or 0.1% trypan blue capsule staining group(193 eyes)and non-stained or placebo staining group(185 eyes)changed without statistical significance \〖Within 1mo after operation(<i>WMD</i>=-10.47, 95% <i>CI</i>=-26.44-5.61, <i>P</i>=0.20); 1mo after operation(<i>WMD</i>=-60.72, 95% <i>CI</i>=-170.92-49.49, <i>P</i>=0.28)\〗. The percentage of corneal endothelial hexagonal cell loss at 1mo after operation changed without statistical significance(<i>WMD</i>=0.50, 95% <i>CI</i>=-2.09-3.09, <i>P</i>=0.71). The central corneal thickness(CCT)at 1mo after operation changed without statistical significance(<i>WMD</i>=3.10, 95% <i>CI</i>=-5.77-11.98, <i>P</i>=0.49). The coefficient of variation(CV)changed without statistical significance(<i>WMD</i>=-1.00, 95% <i>CI</i>=-2.86-0.86, <i>P</i>=0.29).<p>CONCLUSION: 0.02%, 0.06% or 0.1% trypan blue capsule staining in phacoemulsification have no significant effect on the number and function of corneal endothelial cells.

A Meta-analysis of the effect of trypan blue capsule staining on corneal endothelial cells in phacoemulsification / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM:To systematically evaluate the effect of trypan blue capsule staining on corneal endothelial cells in phacoemulsification. <p>METHODS: RCTs on the use of trypan blue for capsular staining in phacoemulsification were retrieved from China Knowledge Network(CNKI), Wanfang Database, Weipu Database, SinoMed, PubMed, SpringerLink, Clinicalkey, Medline, Cochrane Library, Web of Science, OVID, Embase. The search time was from the establishment of the databases to April 2019.The Meta-analysis of the included literature was made by Revman 5.3 and R 3.7.<p>RESULTS: Eight trials included 378 eyes were selected. Meta-analysis showed that the number of corneal endothelial cell loss between 0.02%, 0.06% or 0.1% trypan blue capsule staining group(193 eyes)and non-stained or placebo staining group(185 eyes)changed without statistical significance \〖Within 1mo after operation(<i>WMD</i>=-10.47, 95% <i>CI</i>=-26.44-5.61, <i>P</i>=0.20); 1mo after operation(<i>WMD</i>=-60.72, 95% <i>CI</i>=-170.92-49.49, <i>P</i>=0.28)\〗. The percentage of corneal endothelial hexagonal cell loss at 1mo after operation changed without statistical significance(<i>WMD</i>=0.50, 95% <i>CI</i>=-2.09-3.09, <i>P</i>=0.71). The central corneal thickness(CCT)at 1mo after operation changed without statistical significance(<i>WMD</i>=3.10, 95% <i>CI</i>=-5.77-11.98, <i>P</i>=0.49). The coefficient of variation(CV)changed without statistical significance(<i>WMD</i>=-1.00, 95% <i>CI</i>=-2.86-0.86, <i>P</i>=0.29).<p>CONCLUSION: 0.02%, 0.06% or 0.1% trypan blue capsule staining in phacoemulsification have no significant effect on the number and function of corneal endothelial cells.

Corneal staining during cataract surgery: Natural course, ASOCT features, and preventive measures

We report the natural course of the accidental injection of trypan blue into the corneal stroma while performing a routine cataract surgery by a resident during a training session. The corneal staining resolved with conservative medical treatment over 7 weeks. This case describes the anterior segment optical coherence tomography (ASOCT) features of corneal staining. It emphasizes on the relatively benign nature of this dye and the follow-up course. Causes that may be responsible for this untoward complication are highlighted with the necessary preventive measures that need to be taken care are also discussed.

Investigating the Sonodynamic-Radiosensitivity Effect of Gold Nanoparticles on HeLa Cervical Cancer Cells

BACKGROUND: In this article, we estimated the combined effect of radiotherapy (RT) with ultrasound (US) wave and the ability of gold nanoparticles (GNPs) to improve their combined therapeutic effects. METHODS: At first, HeLa cells received the various treatment modalities: RT (6 MV; 0.5, 1, and 2 Gy), US irradiation (1 MHz; 0.5, 1, and 1.5 W/cm2, 1 minute), and RT+US. Afterwards, the enhanced effect of US on RT was evaluated. Then, the effect of the synthesized GNPs at different concentrations (0.2, 1, and 5 µg/mL, 24 hours) was evaluated to assess the effect on HeLa cells combined with RT+US. Cell survival rates in the different treatment groups at 24, 48, and 72 hours post-treatment were evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and trypan blue assays. RESULTS: Our results show US irradiation could enhance the effect of RT at the same radiation dose and could be utilized as a sensitizer agent for RT. Moreover, our findings indicate RT+US in combination with different nanoparticle concentrations could enhance the effect of RT+US so that they can improve the treatment results up to 9.93 times and act as sonodynamic-radiosensitivity. These results also indicate that the combination of RT with US along with GNPs has synergistic effects compared to RT or US alone. Cell survival results show that combining the low US waves (1.5 W/cm2), GNPs (5 μg/mL), and X-rays (2 Gy) increase the cytotoxicity on HeLa cell up to 95.8%. CONCLUSION: We concluded that GNPs could act as a good sensitizing agent in RT+US irradiation and could result in the synergistic effects.

Evaluation effects of four cytotoxicity methods on in vitro inhibition of shikonin against tumor cells / 中草药

Objective To study the effects of four cytotoxicity evaluation methods on the inhibition rate of shikonin (SK) in vitro, and to compare the pseudo-negative phenomenon often found in the evaluation of cytotoxic activity of natural pigments represented by naphthoquinones in Lithospermum erythrorhizon. Methods SK was co-cultured with its high-sensitive strain HL-60 cells and low-sensitivity strain A549 cells, trypan blue method, sulforhodamine B (SRB) method, Cell Counting Kit-8 (CCK-8) and MTT cytotoxicity test were used for parallel experiments to determine the dose-effect relationship curve of SK (0.4-128 μmol/L) inhibiting the growth of cells. Results The half-inhibitory concentration (IC50) of shikonin on HL-60 cells was determined by trypan blue method, SRB method, CCK-8 method, and MTT method, which was 0.57, 0.77, 1.36, and 1.01 μmol/L, respectively. For A549 cells, the IC50 was 6.30, 10.38, 13.48, and 15.24 μmol/L, respectively. When the concentration of shikonin was below 3.2 μmol/L and 32 μmol/L, the inhibition rate of the two kinds of cells increased linearly by the four methods, followed by differences. Among them, the results of the trypan blue method and the SRB method are in good agreement, while the MTT method and the CCK-8 method have lower inhibition rates. At 12.8 μmol/L, the inhibitory rate of SK on HL-60 cell measured by CCK-8 was 81%, while the inhibitory rate measured by trypan blue method was 96%, and at 128 μmol/L the inhibitory rate of SK on A549 cells measured by MTT method was 89%, however, the inhibitory rate measured by trypan blue method was 99%. The absorption spectrum of SK overlapped with formazan at the wavelength from 400 to 600 nm, with the maximum overlap peak from 550 to 570 nm, and CCK-8 reagent had a synergistic inhibitory effect on HL-60 with SK. The results of trypan blue method showed that SK at the highest dose almost completely killed cells in the plate wells, which was significantly different from the control group, but both MTT method and CCK-8 method resulted in a pseudo-negative phenomenon. Conclusion Therefore, cytotoxicity test of natural pigments represented by naphthoquinones in L. erythrorhizon, MTT method, and CCK-8 method are not recommended, while SRB method and trypan blue method are suggested.

Use of the lower concentration of trypan blue staining technique during cataract surgery / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM: To determine the lowest concentration of trypan blue(TB)used to stain the anterior capsule satisfactorily and to evaluate the clinical significance of trypan-blue-assisted capsulorhexis in cataract surgery. <p>METHODS:Totally 60 cases(60 eyes)of mature age-related cataract, of which the cortex lentis cloudy degree was C4-C5 and the nucleus of the lens was N3, were randomly divided into three groups. Different concentrations of TB, 0.03%, 0.015%, 0.0075%, were used in staining groups respectively to stain the anterior capsule during cataract surgery. All cases were performed with manual small-incision cataract surgery by the same ophthalmologist. The staining effects, the success rate of continuous circular capsulorrhexis(CCC), the posterior capsule rupture and the state of the intraocular lens(IOL)were studied during the operation. The significant statistics was conducted between the groups. The density of corneal endothelial cells, intraocular pressure(IOP), inflammation in anterior chamber, corneal edema, staining of other intraocular structures were also observed at 1d, 1wk, 3mo postoperatively. <p>RESULTS: Trypan blue in concentrations as low as 0.015% stained the anterior capsule satisfactorily, allowing safe creation of a CCC. At concentrations of 0.03% and 0.015%, the success rate of CCC and the state of the intraocular lens(IOL)was significantly better than 0.0075% group, the difference was statistically significant(<i>P</i><0.0083). The difference of posterior capsule rupture rates and the rate of lost corneal endothelial cells in three groups were not statistically significant(<i>P</i>>0.0083). The staining of CCC margin and side port disappeared in 1wk after surgery. Inflammation in anterior chamber of all cases was slight. There was not any evidence of residual stain in the anterior segment during the postoperative period. There was no corneal edema and intraocular pressure higher after 1wk. <p>CONCLUSION: Trypan blue staining of the anterior capsule is a safe and useful technique in cataract surgery, which can raise the success rate of cataract surgery. The 0.015% trypan blue staining, the lowest effective concentration, is strongly considered.

Effect of Trypan Blue on the Survival of Cultured Trabecular Meshwork Cells

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.

Avaliação da composição molecular da cápsula anterior da lente de cães idosos com catarata de alto risco / Molecular evaluation of the anterior lens capsule of older dogs suffering from high risk cataracts

Atualmente, a cápsula anterior e o epitélio da lente tem sido cada vez mais estudados, com o intuito de reduzir as possíveis complicações do pós-operatório da remoção da catarata, tal como a opacidade da cápsula posterior, alteração ocasionada principalmente pela diferenciação e migração das células do epitélio lenticular para a cápsula posterior da lente. O objetivo deste estudo foi analisar a composição molecular da cápsula anterior da lente pela técnica histoquímica de PAS (avaliação de proteoglicanos) e picrosirius red (avaliação de colágeno IV), em cães idosos com catarata diabética e não diabética do tipo hipermadura, submetidos ao uso ou não de azul de tripano a 0,1 % durante a facoemulsificação. Vinte e sete cães foram estudados, incluindo 21 fêmeas e 6 machos, de 8 a 12 anos de idade (média = 9,6 anos), de diversas raças e divididos em 2 grupos: GC (catarata hipermadura) e GCD (catarata diabética). Os resultados das análises realizadas mostraram que ambas as amostras, tanto as provenientes das cataratas hipermaduras, quanto das diabéticas, apresentam semelhante composição molecular de proteoglicanos e colágeno IV e isto independente da utilização de azul de tripano a 0,1 %. Conclui-se, portanto, que se os resultados obtidos forem decorrentes de alterações provocadas pelo rápido metabolismo da catarata diabética e pela cronicidade da catarata hipermadura sugere-se que o comprometimento da estrutura capsular seja de intensidade equivalente e, por consequência, que isto também possa prejudicar o metabolismo das células do epitélio anterior da lente, diminuindo assim a incidência da opacidade da cápsula posterior de cães com catarata diabética e hipermadura submetidos à facoemulsificação.(AU)

Nowadays, the anterior lens capsule and its epithelium have been being frequently studied aiming to reduce the incidence of posterior lens capsule opacity, a complication that frequently occurs after surgical removal of cataracts, due to epithelium cells differentiation and migration to the posterior pole. The objective of this study was to evaluate by histochemistry (PAS and picrosirius red) analysis two important molecular components of the anterior lens capsule (proteoglycans and type IV collagen) in older diabetic and non-diabetic dogs, with diabetic and hypermature cataracts, after phacoemulsification surgery utilizing 0.1% trypan blue or not. Twenty seven dogs, including 21 female and 6 male dogs, with ages varying from 8 to 12 years old (mean = 9.6 yo) of different breeds were studied. The animals were divided into 2 groups: GC (hypermature cataracts) and GCD (diabetic cataracts). Results showed that, besides their different pathophysiologies, both types of capsules studied (diabetic and hypermature ones) presented the same molecular composition of proteoglycans and type IV collagen, since no statistical significant differences were observed. In addition, 0.1% trypan blue was not capable to induce any other evident alteration for the samples. In conclusion, our findings suggest that, if the results consist in alteration induced by the aggressive metabolism of the diabetic cataract or the chronicity of the hypermature one, it is of the same intensity and independent of the use of 0.1% trypan blue. It is also possible to suggest that this alteration must be capable to compromise lens epithelium cell metabolism, which should probably favour future lens posterior capsule studies.(AU)

Avaliação da composição molecular da cápsula anterior da lente de cães idosos com catarata de alto risco / Molecular evaluation of the anterior lens capsule of older dogs suffering from high risk cataracts

Atualmente, a cápsula anterior e o epitélio da lente tem sido cada vez mais estudados, com o intuito de reduzir as possíveis complicações do pós-operatório da remoção da catarata, tal como a opacidade da cápsula posterior, alteração ocasionada principalmente pela diferenciação e migração das células do epitélio lenticular para a cápsula posterior da lente. O objetivo deste estudo foi analisar a composição molecular da cápsula anterior da lente pela técnica histoquímica de PAS (avaliação de proteoglicanos) e picrosirius red (avaliação de colágeno IV), em cães idosos com catarata diabética e não diabética do tipo hipermadura, submetidos ao uso ou não de azul de tripano a 0,1 % durante a facoemulsificação. Vinte e sete cães foram estudados, incluindo 21 fêmeas e 6 machos, de 8 a 12 anos de idade (média = 9,6 anos), de diversas raças e divididos em 2 grupos: GC (catarata hipermadura) e GCD (catarata diabética). Os resultados das análises realizadas mostraram que ambas as amostras, tanto as provenientes das cataratas hipermaduras, quanto das diabéticas, apresentam semelhante composição molecular de proteoglicanos e colágeno IV e isto independente da utilização de azul de tripano a 0,1 %. Conclui-se, portanto, que se os resultados obtidos forem decorrentes de alterações provocadas pelo rápido metabolismo da catarata diabética e pela cronicidade da catarata hipermadura sugere-se que o comprometimento da estrutura capsular seja de intensidade equivalente e, por consequência, que isto também possa prejudicar o metabolismo das células do epitélio anterior da lente, diminuindo assim a incidência da opacidade da cápsula posterior de cães com catarata diabética e hipermadura submetidos à facoemulsificação.

Nowadays, the anterior lens capsule and its epithelium have been being frequently studied aiming to reduce the incidence of posterior lens capsule opacity, a complication that frequently occurs after surgical removal of cataracts, due to epithelium cells differentiation and migration to the posterior pole. The objective of this study was to evaluate by histochemistry (PAS and picrosirius red) analysis two important molecular components of the anterior lens capsule (proteoglycans and type IV collagen) in older diabetic and non-diabetic dogs, with diabetic and hypermature cataracts, after phacoemulsification surgery utilizing 0.1% trypan blue or not. Twenty seven dogs, including 21 female and 6 male dogs, with ages varying from 8 to 12 years old (mean = 9.6 yo) of different breeds were studied. The animals were divided into 2 groups: GC (hypermature cataracts) and GCD (diabetic cataracts). Results showed that, besides their different pathophysiologies, both types of capsules studied (diabetic and hypermature ones) presented the same molecular composition of proteoglycans and type IV collagen, since no statistical significant differences were observed. In addition, 0.1% trypan blue was not capable to induce any other evident alteration for the samples. In conclusion, our findings suggest that, if the results consist in alteration induced by the aggressive metabolism of the diabetic cataract or the chronicity of the hypermature one, it is of the same intensity and independent of the use of 0.1% trypan blue. It is also possible to suggest that this alteration must be capable to compromise lens epithelium cell metabolism, which should probably favour future lens posterior capsule studies.

Evaluation of Cytotoxic Activity of Sargassum vulgare From the Lebanese Coast Against Jurkat Cancer Cell Line.

The discovery of cancer drugs that effectively destroy cancer cells or stop their growth without toxicity to normal cells is a challenge to enhance the therapeutic effects and reduce side effects. Many papers have highlighted the implication of marine algae that show anticancer activity. In this report, we assessed the cytotoxic activity of two different crude extracts from Sargassum vulgare (Sargassaceae), a marine brown algae collected from the Lebanese coast, against Jurkat human cancer cell line using trypan blue exclusion test. Both extracts, water: ethanol extract and chloroform: ethanol extract, showed cytotoxic activity against Jurkat cancer cell line with IC50 values of 136.907 μg/ mL and 49.056 μg/ mL, respectively after 72 hours of treatment. Further research designed to isolate and identify novel and efficient anticancer drug candidates from these seaweed extracts need to be explored.

Application of trypan blue and indocyanine green in mature and hypermature cataracts / 国际眼科杂志(Guoji Yanke Zazhi)

Abstract?AlM: To assess the efficacy and safety of trypan blue and indocyanine green ( lCG ) for continuous curvilineal capsulorrhexis ( CCC ) in mature or hypermature phacoemulsification.?METHODS: A total of 122 eyes of 122 cases with cataracts in mature and hypermature were randomly divided into three groups, trypan blue staining group was 46 eyes of 46 cases as group A, lCG staining group was 40 eyes of 40 cases as group B, control group was 36 eyes of 36 cases as group C. Staining groups were used to 0. 2mL trypan blue or lCG injected into the anterior chamber during operation, respectively. The success rate of CCC, lens posterior capsule rupture and implanted intraocular lens pouch were observed and compared during operation. Anterior chamber inflammation was observed after operation, and compared with the control group to observe and analysis.? RESULTS: The success rate of CCC, implanted intraocular lens pouch were statistically significant difference in trypan blue staining group ( group A ) than that in control group (group C) (P 0. 05 ) . Anterior chamber inflammation was no significant difference in the postoperative reaction among the three groups.?CONCLUSlON:The application of trypan blue or lCG for lens capsule staining before CCC in lack of red reflective mature and hypermature cataracts is safe and effective, both results are comparable. lt guarantees a complete CCC and improves the success rate of phacoemulsification.

Evaluation of the safety of intracameral trypan blue injection on corneal tissue using oxidative stress parameters and apoptotic activity: an experimental study / Avaliação da segurança de injeção de azul de tripan intracameral no tecido da córnea utilizando parâmetros de estresse oxidativo e atividade apoptótica: um estudo experimental

Purpose: The present experimental study aimed to investigate the effects of intracameral trypan blue (TB) on oxidative stress parameters and apoptosis in corneal tissue. Methods: Thirty rats were randomly assigned to three groups of 10 rats each: the sham group (Group 1); control group (Group 2); and treatment group (Group 3). The control group was administered 0.01 cc of balanced salt solution. The treatment group was administered 0.006 mg/0.01 cc of TB. The total antioxidant status (TAS) and total oxidant status (TOS) in corneal tissue and blood were measured and the oxidative stress index (OSI) was calculated. Finally, corneal tissue histopathology was evaluated using staining for caspase-3 and -8, and apoptotic activity was examined. Results: The TAS, TOS and OSI levels in the blood samples were not significantly different (p>0.05 for all). Compared with the sham and control groups, the TOS and OSI levels in corneal tissue were significantly different in the treatment group (p<0.05 for all). No significant difference was observed between the sham group and the control group (p>0.05). Immunohistochemical staining for caspase-3 and caspase-8 demonstrated higher apoptotic activity in the TB group than in the sham and control groups. Conclusion: The present study showed that intracameral TB injection is safe systematically but may be toxic to corneal tissue, as demonstrated using oxidative stress parameters and histopathological evaluation. .

Objetivo: Este estudo experimental tem como objetivo investigar os efeitos do azul de tripan intracameral (TB) sobre parâmetros de estresse oxidativo e apoptose no tecido da córnea. Métodos: Trinta ratos foram divididos aleatoriamente em três grupos de 10 animais cada: grupo simulação (Grupo 1); grupo controle (Grupo 2); e grupo tratamento (Grupo 3). No grupo controle foi administrado 0,01 cc de solução salina balanceada (BSS). No grupo tratamento foi administrado 0,006 mg/0,01 cm de TB. O estado antioxidante total ( TAS) e estado oxidante total ( TOS) no tecido da córnea e sangue foram medidos e o índice de estresse oxidativo (OSI) foi calculado. Finalmente, histopatologia do tecido da córnea foi avaliada por meio da coloração para caspase-3 e -8; atividade apoptótica também foi examinada. Resultados: Os níveis de TAS, TOS e OSI das amostras de sangue não foram significativamente diferentes (p>0,05 para todos). Em comparação com os grupos simulação e controle, os níveis de TOS e OSI no tecido da córnea foram significativamente diferentes no grupo tratamento (p<0,05 para todos). Não houve diferença significativa entre o grupo simulção e o grupo controle (p>0,05). A coloração imuno-histoquímica com a caspase-3 e caspase-8 demonstrou maior atividade apoptótica no grupo tratamento do que nos grupos controle e simulação. Conclusão: Este estudo mostrou que a injeção intracameral TB é segura sistematicamente, mas pode ser tóxica ao tecido da córnea, como demonstrado através de parâmetros de estresse oxidativo e avaliação histopatológica. .

Trypan blue exclusion assay by flow cytometry

Dye exclusion tests are used to determine the number of live and dead cells. These assays are based on the principle that intact plasma membranes in live cells exclude specific dyes, whereas dead cells do not. Although widely used, the trypan blue (TB) exclusion assay has limitations. The dye can be incorporated by live cells after a short exposure time, and personal reliability, related to the expertise of the analyst, can affect the results. We propose an alternative assay for evaluating cell viability that combines the TB exclusion test and the high sensitivity of the flow cytometry technique. Previous studies have demonstrated the ability of TB to emit fluorescence when complexed with proteins. According to our results, TB/bovine serum albumin and TB/cytoplasmic protein complexes emit fluorescence at 660 nm, which is detectable by flow cytometry using a 650-nm low-pass band filter. TB at 0.002% (w/v) was defined as the optimum concentration for distinguishing unstained living cells from fluorescent dead cells, and fluorescence emission was stable for 30 min after cell treatment. Although previous studies have shown that TB promotes green fluorescence quenching, TB at 0.002% did not interfere with green fluorescence in human live T-cells stained with anti-CD3/fluorescein isothiocyanate (FITC) monoclonal antibody. We observed a high correlation between the percentage of propidium iodide+CD3/FITC+ and TB+CD3/FITC+ cells, as well as similar double-stained cell profiles in flow cytometry dot-plot graphs. Taken together, the results indicate that a TB exclusion assay by flow cytometry can be employed as an alternative tool for quick and reliable cell viability analysis.

Anticancer Properties of Novel Rhenium Pentylcarbanato Compounds against MDA-MB- 468(HTB-132) Triple Node Negative Human Breast Cancer Cell Lines.

Aim: To study the efficacy of novel rhenium compounds to treat triple node negative breast cancer. Study Design: Six (6) novel rhenium pentycarbanato compounds (PC1-6) were synthesized and triple node negative breast cancer cell lines HTB-132 and Balb/c mouse kidney cell lines were treated with each of them for 48 hours. The results were analyzed by a common trypan blue cell death assay system and statistically analyzed. Place and Duration: The compounds were synthesized, analyzed and evaluated at the Department of Chemistryof Morgan State University, Baltimore, Maryland and the Pharmaceutical Sciences Department of Elizabeth City State University campus of the University of North Carolina system. Methodology: The novel rhenium compounds were synthesized from one-pot reactions of Re2(CO)10 with the corresponding α-diimine ligands in 1-pentanol.The compounds were characterized spectroscopically. The cell lines were cultured by standard cell culture procedure and treated with each of the six compounds in DMSO for 48 hours with a negative control and a DMSO vehicular control along with a cisplatin positive control.The cytotoxicity was evaluated by standard trypan blue assay and the results were statistically analyzed. Results: The trypan blueassay reveals that these compounds have significant cytotoxicity against MDA-MB-468 (HTB-132) triple node negative breast cancer cell lines and are less nephrotoxic than cisplatin. Conclusion: The novel rhenium compounds PC 1-6 can potentially find applications in the treatment of highly malignant triple node negative breast cancer.

Anticancer activity of Sargassum wightii Greville on Dalton's ascitic lymphoma / 中国天然药物

AIM@#Sargassum wightii Greville is a marine brown alga belonging to the Sargassaceae family which has about 200 species. The ethanolic extract of the whole dry plant powder contained numerous phytoconstituents, including flavonoids. The study was focused on the anticancer activity of Sargassum wightii in mice.@*METHOD@#The ethanolic extract of Sargassum wightii (EESW) at two dose levels was used to examine the anticancer activity in mice using DAL cell lines to induce cancer. The body weight, viable and non-viable tumor cell count, mean survival time, increase in life span, and hematological parameters were observed for anticancer activity of EESW.@*RESULTS@#The intraperitoneal inoculation of DAL cells in mice significantly increased cancer cell count. The decrease in the cancer cell number observed in the EESW-treated group cancer animals indicates that the test drug has a significant inhibitory effect on the tumor cell proliferation. Treatment with EESW also showed a significant decrease in tumor weight, and hence increased the lifespan of DAL-treated mice. In addition, EESW administration significantly restored the hematological parameters in DAL-treated mice.@*CONCLUSION@#The present study results suggest that administration of extract offers enhanced antioxidant potential. Therefore it can be concluded from this study that EESW possesses anticancer activity.

Assessment of Cell Viability in Umbilical Cord Blood by Using 7-AAD/annexin V Dual Staining

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.

Histopathologic study of lens epithelial cells in different types of cataracts / 中华实验眼科杂志

Background Cataract was directly associated with the damage to the structure and function of lens epithelial cells (LECs).In those patients who suffer from cataracts,morphologic changes of LECs is the most compelling evidence confirming loss of cellular structure and function of LECs.So,learning about the morphological changes of LECs of the different types of cataracts is very important for study on biological behaviors of LECs in different environments or diseases.Objective This study was to evaluate the morphological and ultrastructural changes of the LECs in different types of cataracts.Methods Anterior capsular member from age-related cataracts,diabetic cataracts and high myopia complicated cataract were obtained during the cataract surgery and 15 pieces for each.Trypan blue and alizarin red (TB-AR) stain,haematoxylin and eosin stain were performed in the samples to assess the viability and morphology of LECs.The ultrastructural change of LECs was observed under the transmission electron microscope.The features of the LECs were compared among the different types of cataract.Results TB-AR stain showed that LECs were polygon in shape with the mosaic arrangement and round cell nucleus,and a few dead cells were seen in the samples age-related cataract.In the diabetic samples,LECs largened from swelling with different sizes.More dead cells were found in the high myopia complicated cataract.Haematoxylin and eosin stain exhibited that the anterior capsular membrane presented a homogeneuous membrane,and monolayer LECs attached firmly anterior capsular membrane in the samples of related cataract.Majority of the cells had the intact structure.However,the interspaces between cells and capsular membrane were found in diabetic cataract.Also,smaller LECs were seen in high myopia complicated cataract with the irregular cell morphology.Under the transmission electron microscope,LECs presented the normal shape,intact intercellular tight junction and well attachment between cells and capsular membrane in the samples of the age-related cataract.In the samples of the diabetic cataract,edema of LECs and large intercellular spaces were seen.In addition,the jogged pump and vacuolar degeneration of cytoplasm were revealed in the high myopia complicated cataract.Conclusions The degeneration,necrosis and apoptosis was a common pathological basis of age-related cataract,diabetic cataract and high myopia complicated cataract.However,the damage of LECs was more serious in diabetic cataract and high myopia complicated cataract than that of agerelated cataract.

Trypan Blue Dye as an Adjunct to Cataract Surgery.

The current study was done to evaluate the utility and safety of Trypan Blue staining of the anterior capsule for enhancing visualization of capsulorrhexis in mature and hypermature cataracts.This study included 100 eyes of 100 patients with a unilateral mature or hypermature cataract. In all these cases 0.2ml of 0.1% Trypan blue dye was used to stain the anterior capsule in cataract surgery. In all 100 eyes the Continuous Curvilinear Capsulorrhexis (CCC) was completed. Successful cataract surgery with intraocular lens (IOL) implantation was performed in all eyes. Adverse reactions related to the dye such as raised intraocular pressure or anterior chamber inflammation was not observed in the immediate postoperative period or at the end of mean follow-up of 3 months.Trypan blue dye staining of the anterior capsule was found to be an effective and safe technique that helps in completion of Continuous Curvilinear Capsulorrhexis (CCC) in mature and hypermature cataracts.

Progressive atrophy of retinal pigment epithelium after trypan-blue-assisted ILM peeling for macular hole surgery.

We report a case of progressive atrophy of the retinal pigment epithelium (RPE) after trypan-blue-assisted peeling of internal limiting membrane (ILM) for macular hole surgery. A 68-year-old Caucasian female underwent a 20-g pars plana vitrectomy for a chronic stage-3 macular hole. The ILM was stained with 0.06% trypan blue (VisionBlue™, DORC Netherlands) for 2 min after fluid air exchange. Dye was reapplied for another 2 min due to poor staining. The ILM was completely removed around the macular hole with forceps. RPE atrophy was noticed at the edge of the hole 1 month after surgery. It progressively increased in intensity and enlarged over 2 years. Her final visual acuity was counting fingers, significantly worse compared to her presenting visual acuity of 20/200. Progressive atrophy of RPE in our patient was most likely due to the toxicity of trypan blue. Reapplication of the dye may increase the likelihood of toxicity.