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Fluorescein isothiocyanate-labeled insulin (FITC-insulin) has been widely used for bioanalytical appli-cations. Due to the high cost of commercial FITC-insulin and tedious labeling procedures described in the literature, there is still a need to develop a cost effective, reliable and quick labeling method for insulin. The purpose of the present work was to develop a quick and affordable method for FITC labeling of human insulin and to determine the effect of different conjugations of FITC to human insulin on its permeability through the MDCK cell monolayer. FITC labeling of insulin gives mono-, di-or tri-conjugates depending on the reaction time and the molar ratio of FITC:insulin. Mono-conjugate with unlabeled insulin, mixture of di-and tri-conjugate, and tri-conjugate with very little amount of di-conjugate were synthesized in less than 4 h. Degree of conjugation had an effect on the permeability of insulin through the MDCK cell monolayer. Mono-conjugate had higher permeability than the unlabeled insulin due to increase in partition coefficient. However, tri-conjugate showed lower permeability than the unlabeled insulin due to the increase in molecular weight.
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Aim To reveal the effects of Guizhi decoction (G Z D) on allergic contact dermatitis by establishing the mouse allergic contact dermatitis model induced by fluoresciniso thiocyate (FITC) and culturing splenic lymphocytes. Methods BALB/c mice were sensitized by smearing the 1.5% FITC on the shaved abdomens on day 1 and day 2 and elicited on the right ear with 0. 6% FITC solution on day 6. GZD was administered from day 1 to day 7. Ear thickness was measured 24 h after elicitation. HE staining was performed for pathohistological examination. Interleukin-4 (I L - 4), IL-5, IL-9, interferon γ(IFN-γ) in ear tissue homogenates and IgE in serum were detected. The splenic lymphocytes were stimulated with ConA. Results The ear swelling was significantly suppressed by administering GZD. GZD reduced the infiltration of inflammatory cells and edema in the ear, and the levels of I L 4, IL-9, IFN7 and IgE. GZD could also reduce the expression of I L 4, IL-10, IFNγ and GATA3. Conclusions GZD could suppress allergic contact dermatitis elicited by FITC, which may result from inhibiting the differentiation of lymphocytes to Th2 cells.
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OBJECTIVES@#To establish an experimental method for evaluating material permeability of type I collagen hydrogels.@*METHODS@#Using BSA-FITC as an indicator, by combining BSA-FITC with PBS they were used as permeability media, and using transwell load hydrogen sample to detect BSA-FITC transparent rate.@*RESULTS@#In the concentration range of 100 μg·mL~0.781 μg·mL, the standard curve ≥ 0.99, Lower Limit of Quantity (LLOQ) is 3.125 μg·mL, RSD <5%, detection recovery rate is in the range of 80%~120%.@*CONCLUSIONS@#In this study, we established an experimental method for evaluating material permeability of hydrogel. The BSA-FITC transparent rate of type I collagen hydrogel was 100% at 28 h.
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Collagen Type I , Chemistry , Hydrogels , Chemistry , Materials Testing , PermeabilityABSTRACT
The objective of this work is to evaluate the effect of polyamidoamine (PAMAM) dendrimers on electroosmotic flow (EOF) through skin. The effect of size and concentration of dendrimer was studied, using generation 1, 4 and 7 dendrimer (G1, G4 and G7, respectively). As a marker molecule for the direction and magnitude of EOF, a neutral molecule, acetoaminophen (AAP) was used. The visualization of dendrimer permeation into the current conducting pore (CCP) of skin was made using G4–fluorescein isothiocyanate (FITC) conjugate and confocal microscopy. Without dendrimer, anodal flux of AAP was much higher than cathodal or passive flux. When G1 dendrimer was added, anodal flux decreased, presumably due to the decrease in EOF by the association of G1 dendrimer with net negative charge in CCP. As the generation increased, larger decrease in anodal flux was observed, and the direction of EOF was reversed. Small amount of methanol used for the preparation of dendrimer solution also contributed to the decrease in anodal flux of AAP. Cross-sectional view perpendicular to the skin surface by confocal laser scanning microscope (CLSM) study showed that G4 dendrimer-FITC conjugate (G4-FITC) can penetrate into the viable epidermis and dermis under anodal current. The permeation route seemed to be localized on hair follicle region. These results suggest that PAMAM dendrimers can permeate into CCP and change the magnitude and direction of EOF. Overall, we obtained a better understanding on the mechanistic insights into the electroosmosis phenomena and its role on flux during iontophoresis.
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Acetaminophen , Dendrimers , Dermis , Electroosmosis , Epidermis , Fluorescein-5-isothiocyanate , Hair Follicle , Iontophoresis , Methanol , Microscopy, Confocal , SkinABSTRACT
To prepare the asiaticoside nanoemulsions (ASI-NEs) and asiaticoside nanoemulsions-based gels (ASI-NBGs), compare them with the commercial cream of asiaticoside (ASI-C) in terms of transdermal characteristics, and investigate the transdermal mechanism of ASI-NEs and ASI-NBGs. Their transdermal characteristics were studied by using Franz diffusion cells. The effect of topical ASI-NEs and ASI-NBGs on ultrastructure of rabbit skin was evaluated by using HE staining method. The localization and the permeation pathway of asiaticoside were visually investigated by using laser scanning confocal microscope (CLSM). The transdermal studies in vitro showed that the cumulative amount of ASI permeated from ASI-NEs and ASI-NBGs at 12 h after application were (3 504.30±180.93), (1 187.40±128.88) μg·cm⁻² respectively, 6.57, 2.23 times of that in the control group of ASI-C; the drug deposition of ASI-NEs and ASI-NBGs in skin was (159.48±7.47), (120.53±5.71) μg·cm⁻² respectively, 5.93, 4.48 times of that of ASI-C. HE staining of the rabbit skin after application of ASI-NEs and ASI-NBGs showed that the epidermis structure was basically intact; stratum corneum was loosed and the keratin fragment was increased; at the same time, the gap of prickle cell was increased and the basal cells were arranged loosely. The study of CLSM showed that significant percutaneous enhancer effect was observed for ASI-NEs after the topical application of 6 h, as the fluorescent compound was penetrated in the dermis and diffused uniformly. The fluorescence area and the integral optical density (IOD) were 28.81, 32.51 times of that in the FITC aqueous solution group, respectively. The fluorescent preparations showed strong fluorescence in the epidermis, but weak in deeper layers; with the increase of treatment time, the fluorescence in deeper layer was increased and stronger in skin appendages. The prepared ASI-NEs and ASI-NBGs have good transdermal characteristics and the transdermal mechanism is related to breaking the ultrastructure of stratum corneum and penetrating by the path of skin adnexa.
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AIM To investigate the tissue distribution of brucine-loaded solid lipid nanoparticles in mice in vivo.METHODS Mice were intravenously injected with suspension of prepared brucine-loaded solid lipid nanoparticles and marked by fluorescein isothiocyanate (FITC).The in vivo tissue distribution of nanoparticles was analyzed by having the brucine contents in various tissues (heart,liver,spleen,lung,kidney and bone) determined by HPLC,after which fluorescence confocal laser endomicroscopy was used for further detection.RESULTS Brucine had its the highest (1.64) relative intake efficiency (Re) in mice liver,and the nanoparticles shared all over one value of targeting efficiencies (Te) in various tissues,manifesting a much stronger selectivity to liver than that of brucine solution.With the extension of time,the FITC-narked nanoparticles displayed a rich extracellular to intracellular distribution indicating a positive correlation.CONCLUSION Brucine's increased distribution in the liver tissue of mice due to its solid lipid nanoparticle form shows obvious for liver targeting.
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Objective:To establish an HPLC-FD method for determining the content of Angelica sinensis polysaccharide (ASP1) to lay foundation for its pharmacokinetic study in rats. Methods: Purified ASP1 was labeled with FITC by the method of Belder and Granath to obtain ASP1-FITC. The tissue samples were treated with 30% trichloroacetic acid and 11% NaOH before injection. The samples were determined by HPLC-FD. A PL aquagel-OH MIXED column was used,and the mobile phase was phosphate buffer( dis-solve NaH2PO32. 34g , Na2HPO34. 33 g and NaCl 11. 70 g into 1000 ml water) with pH of 7. 0. The flow rate was 0. 5 ml·min-1. The excitation and emission wavelengths was set at 495 nm and 520 nm, respectively. Results:The linear calibration curve was within the concentration range of 0. 25-40. 00 μg·ml-1(r=0. 9996)with the lower limit of quantification of 0. 20μg·ml-1 in tissue sam-ples. The extraction recovery of ASP1 was determined at low, medium and high concentration with the recovery of 91. 98%-114. 20%. The intra and inter-day RSDs were lower than 8. 31% and 2. 94%, respectively. Conclusion:The method to determine the content of ASP1 in rats by HPLC-FD with pre-column derivatization has been esablished. It is simple, accurate and reliable, which can be suc-cessfully applied in the study of pharmacokinetics and tissue distribution of ASP1 in rats.
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Objective To prepare anti-folic acid (FA) polyclonal antibody and develop a new non-balanced competing chemiluminescence analysis for clinical detection of FA.Methods Established the detection method by added FITC-FA-analogs and FAHRP-antibody in the light emitting plate,which coated with anti-FITC antibody,to form the immune response complex of FITC/antibody-FITC-FA-analogs/FA-antibody-HRP.Then methodology evaluation was performed to evaluate the method performance;and further compared the detecting results with non-FITC system detection system and Electrochemiluminescence system (Roche Elecsys 2010).Results The ELISA results showed that the prepared anti-FA antibodies can recognize serum FA specificly.The methodology evaluation indicated that the linear correlation coefficient of the standard curve was 0.990 0;the analytical sensitivity was 1.21 ng/mL;the range of linear detection was 1.21～ 38.80 ng/mL;The coefficient variability of intra-assay was ＜5 ％,which was better than the results of non-FITC detection system;and the correlation coefficient was 0.908 1 compared with the Elecsys-2010 detection system.Conclusion The established chemiluminescence immunoassay for human serum FA has a good sensitivity and specificity,and suitable for clinical serum FA quantitativedetecting.
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Objective To investigate the possible pathway of FITC-dextran to the cochlea after post-aural injection.Methods The FITC-dextran(weight between 3 000~5 000) was chosen as a tracer in this study.A total of 200 suckling mice were randomly divided into four groups, with 50 in each group.Each animal was then administered with FITC-dextran or dextran via either post-auricular or intra-muscular injection, to a total dose of 20 μl (5 mg/ml).Samples were obtained at 0, 1/12, 1/4, 1/2, 1, 3, 5, 7, 12, and 24 hours after adminstmiceion, and the confocal technique was used to observe the distribution of the tracer.Taking into consideration the influence of spontaneous fluorescence, the fluorescence intensity ratio of the experimental and control groups was used as the final statistical data.Results FITC-dextran injected intramuscularly group: The fluorescence signal can be detected in the sigmoid sinus(SS) 3h after management, while in endolymphatic sac and cochlea at 12 h.FITC-dextran injected post-aurally group: After administration, an obvious fluorescence signal could be observed in the sigmoid sinus and endolymphatic sac immediately, cochlea at 30 min.The signal of the sigmoid sinus, endolymphatic sac and cochlea gradually increased successively, peaked at 5~15 min, 30 min and 60 min, and then decreased gradually.At 12 h, another small increases appeared, and the signal could not be detected at 24 h.Conclusion Compared with intramuscularly application, post-auricular injection can allow the drug to directly reach the endolymph.It is possible that the tracer first gathered in the SS via local blood circulation or infiltration, then entered the ES via micro-circulation around, and eventually arrived at the cochlea.
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Objective: To prepare foliate-conjugated chitosan nanoparticles loaded with berberine hydrochloride (BH/FA-CTS-NPs) and investigate the optimizing technology, physicochemical characterizations, and inhibitory effect on CNE-1. Methods: Folate-conjugated chitosan (FA-CTS) was prepared by amino reaction of folic acid active ester and chitosan molecules; BH/FA-CTS-NPs were prepared using ion cross-linking technique with BH as a model drug. The morphology, particle size, and physicochemical characteristics such as entrapment efficiency (EE), drug-loading, and release in vitro of nanoparticles were studied. The effect of cell anti-migratory and anti-invasive actions of BH/FA-CTS-NPs was investigated using MTT assays, wound healing assays and Annexin-V-FITC single staining assays, respectively. Results: The prepared BH/FA-CTS NPs were round, and the size uniformity adhesion was not found. The results of mean particle size, EE, and drug-loading amount were (282.4 ± 4.5) nm, (89.82 ± 2.91)%, and (9.16 ± 1.01)%, respectively. (80.32 ± 3.24)% of BH in nanoparticles was released within 5 h and then released slowly, and the accumulative release rate within 24 h was (90.92 ± 5.21)%. These results by MTT assay and wound healing assay indicated that BH/FA-CTS NPs not only inhibited the proliferation of CNE-1 cells in a concentration- and time-dependent manner, but can induced apoptosis. Conclusion: BH/FA-CTS NPs with the sustained release effect could be prepared successfully by the ionic crosslinking method. Considering these properties, block proliferation and impair the migration of the CNE-1 cell line, BH/FA-CTS NPs could be an important compound for consideration in the treatment of nasopharyngeal carcinoma.
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Aim To investigate the effects of astragalo-side IV on apoptosis of PC12 cells inducedby hypoxia/hypoglycemia and reoxygenation. Metheds PC12 cells were randomly divided into 4 groups: normal control group,hypoxia/hypoglycemia and reoxygenation group, astragaloside Ⅳ group and vehicle group. Hypoxia/hy-poglycemia and reoxygenation group, astragaloside Ⅳgroup and vehiclegroup were exposed to reoxygenation (12 h) after 3 h of oxygen and glucose deprivation, and astragaloside Ⅳ was added into cells at the same time. Inverted microscope was used to observe the morphological changes ofPC12 cells and MTT method to detect the activities of PC12 cells, and Annexin V-FITC/PI assay and TUNEL staining were used to meas-ure the apoptosis of PC12 cells. Results Compared with normal control group, cells became round or swol-len and its cellula processes were retracted or disap-peared in hypoxia/hypoglycemia and reoxygenation group;a large number of apoptotic cells could also be observed,whose nucleus were shrinkaged, fragmented or deep-stained. The activities of hypoxia/hypoglycemia and reoxygenation group were decreased markedly than those in normalcontrol group(P0. 05 ) . Conclusion Astragaloside IV can reduce the damage of PC12 cells induced by hypoxia/hypoglycemia and reoxygenation, increase cell activity and inhibit cell apoptosis.
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Objective: To prepare neurotoxin nanoparticles (NT-NP) labeled by FITC and modified by polysorbate-80 (P-80), and to compare the in vivo tissue distribution after intranasal administration in rat. Methods: The FITC labeled P-80-NT-NP and NT-NP were prepared, blood samples were collected after intranasal administration in rats at 5, 15, 30, 60, 120, and 240 min, and the brain, heart, liver, spleen, lung, kidney tissue samples were collected. Using FITC-NT as the index component and fluorescence analysis method, the differences of the nanoparticles distribution in rats tissues after modified by P-80 were investigated. Results: After 5, 15, 30, 60, 120, and 240 min of intranasal administration of P-80-NT-NP and NT-NP, the drug was distributed in plasma, heart, liver, spleen, lung, kidney, and brain. After 120 min of administration, the NT concentration in plasma and brain of P-80-NT-NP group was higher than that of NT-NP group, while the NT concentration in liver was lower than that in NT-NP group, with significant difference. Conclusion: P-80 can effectively increase the nasal absorption of NT-NP into the brain and other tissues, which provides the basis for further development and application of preparation of the drug targeting to brain.
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Objective To construct recombinant eukaryotic expression vector for EGFP fused HIV-1 integrase expression. Methods Wild type of HIV-1 integrase gene was cloned into eukaryotic expression vector-pcDNA6/V5-HisA. After restricted enzyme mapping, PCR confirmation and sequence confirmation, the recombinant plasmid was transfected into HeLa cells with Lipofectamine2000. After 24 hours, the expression of integrase was examined by immunofluorescence with confocal fluorescent microscopy. The cells were fixed with 4%paraformaledhyde. The cell nuclei were stained with Propidium Iodide (PI). Then the expression was imaged and analyzed with Confocal Microscopy. Results The integrase expressed significantly in HeLa cells in 24 hours after transfection. Integrase was expressed and localized into nuclei mainly. After fixed with 4% paraformaldehyde, the cell nuclei were stained with PI. When nuclei were showed in red in normal cells, the nuclei with integrase over expression turned yellow or orange. Conclusion The construction of eukaryotic expression vector of integrase was successful. Integrase was expressed and localized into nuclei mainly after transfection in HeLa cells.
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Objective To Investigate the in vitro and in vivo release of chitosan-alginate microgels coated layer-by-layer by polyelectrolyte self-assembly. Methods The cores of the microgels were made by gelatinization using electrostatic microencapsulated and coated by polyelectrolytes using electrostatic attraction. The effects of different layers and ratios of polymer on the in vitro lease of FITC-dextran were evaluated. Histrological examination was carried out to observe the in vivo release process by injecting the coated microgels into mice. Results The results showed that alginate and calcium chloride concentrations and polyelectrolyte layers markedly affected the lag time of pulsed release and the relasing speed after lagging. Conclusion The release of microgels coated layer-by-layer by polyllectrolyte can be controlled in vitro and can be observed in vivo; meanwhile, the microgels are safe and have good biocompatibility.
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Objective To detect the expression level of human zinc finger 23 (ZNF23) in hepatocellular carcinoma tissue samples and HepG2 cell lines and investigate the relationship between hZNF23 expression and clinicopathological characteristics of HCC and cell apoptosis. Methods The expression levels of hZNF23 and GAPDH mRNA in 37 cases of HCC were measured by real-time RT-PCR. The association between the expression of hZNF23 and the clinicopathological characteristics of HCC was analyzed. Cultured HepG2 cells were divided into 4 groups ( control group, 1.25 μg/ml , 2.5 μg/ml and 5 μg/ml cisplatin)or 6 groups( control group, 1.25 μg/ml, 2.5 μg/ml, 5 μg/ml, 10 μg/ml and 20 μg/ml cisplatin). MTT method was employed to evaluate cell proliferation. Annexin V-FITC assay was used to assess percentage of apoptotic HepG2 cells. The expression levels of hZNF23 and GAPDH mRNA of HepG2 cells after apoptosis induced by cisplatin with a series of concentrations were measured by real-time RT-PCR.Results The median ( quartile1, quartile 3) expression levels of hZNF23 mRNA in 37 HCC tissue samples and adjacent tissue samples were 8.84 (3.59-15.05), 22.20 ( 13.85-42.90 ), respectively. There was significant difference ( U = 259.5, P ＜ 0.01 ). The median ( quartile1, quartile 3 ) expression levels of hZNF23 mRNA in cancer tissue samples with Edmondson stage Ⅰ + Ⅱ [12.80(4.80-19.50)] was much higher than those in stage Ⅲ + Ⅳ [5.01 ( 2.88-11.68 ), U = 99.00, P ＜ 0.05] The median ( quartile1,quartile 3 ) expression levels of hZNF23 mRNA in patients with and without hepatic cirrhosis were 9.92(3.80-15.25) , 3.21 (2.78-3.60), respectively. The median ( quartile1, quartile 3 ) expression levels of hZNF23 mRNA in HBV infection and non-infection patients was 9.09(3.72-15.25 ), 2.48 (1.79-12.10),respectively. There was no significant difference between groups with and without hepatic cirrhosis and between HBV infection and non-infection groups( U = 16. 00 and 24.00, P ＞0.05 ). MTT assay indicated that cisplatin significantly inhibited HepG2 cells proliferation in a dose-dependent manner. Annexin V-FITC/PI assay showed that HepG2 cells apoptosis rates were (0.9 ± 0.2 ) %, ( 4. 2 ± 0.3 ) %, ( 9.8 ± 4. 3 ) %,(23.0 ± 6.0)%, respectively. Cisplatin significantly induced HepG2 cells apoptosis in a dose-dependent manner( F = 27.89, P ＜ 0.01 ). The expression levels of hZNF23 mRNA in cisplatin groups [( 10.39 ±3.08) × 10-5, (24.10 ± 2.09) × 10-5, (6.90 ± 2.24) × 10-4] were significantly lower than that of the controlgroup[(94.80±1.80) ×10-5, F=6.027, P＜0.01]. Conclusions The expression level hZNF23 mRNA is related to Edmondson stage of HCC. The apoptosis effect of cisplatin on HepG2 cells may be associated with the upregulation of hZNF23.
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Em meados da década de 50 iniciou-se o desenvolvimento da citometria de fluxo, tecnologia que permite verificar características físico-químicas de células ou partículas suspensas em meio fluido. Esta tecnologia utiliza anticorpos monoclonais marcados com fluorocromos como ferramenta de investigação em diversas análises e necessita de controles isotípicos para definição da região negativa (background). Estes controles são constituídos por imunoglobulinas de mesmo isotipo e fluorocromo dos anticorpos testes, sendo o isotiocianato de fluoresceína (FITC) o marcador fluorescente mais utilizado na conjugação de anticorpos. Os controles isotípicos têm como função definir a fluorescência inespecífica (células negativas) e as regiões fluorescentes (células positivas). No presente estudo foi selecionado anticorpo monoclonal murino (AcMm) dirigido contra antígeno eritrocitário canino, produzido no Laboratório de Anticorpos Monoclonais do Hemocentro de Botucatu, o qual reage positivamente com hemácias de cães, mas nunca com leucócitos humanos, tendo, portanto, potencial utilidade como controle negativo em citometria de fluxo. A purificação do AcMm da subclasse IgG1 foi feita por cromatografia de afinidade em Proteína-A Sepharose, e o controle da purificação realizado por eletroforese em géis de ágarose e poliacrilamida (SDS-PAGE). A imunoglobulina purificada foi conjugada ao FITC e filtrado em coluna de Sephadex G-25 para separação das proteínas marcadas e não-marcadas. O AcMm conjugado foi testado contra hemácias de cães, e o êxito da conjugação comprovado por testes de fluorescência, sendo a mediana de positividade de 94,70. Frente a leucócitos humanos a mediana de positividade foi 0,03 contra 0,50 dos reagentes comerciais. Os testes estatísticos não-paramétricos de Wilcoxon e correlação de Spearman comprovaram a eficiência e validam o controle isotípico produzido em comparação aos reagentes comerciais testados.
It was during the 1950's that the development of flow cytometry started, technology that allow to measure physiochemical characteristics of cells or suspended particles in fluid. This technology uses monoclonal antibodies labeled by fluorochromes as investigation tool in several analysis and needs isotype controls to define the negative region (background). These controls are constituted by immunoglobulins of the same isotype and fluorochrome from test antibodies, being fluorescein isothiocyanate (FITC) the most fluorescent marker used in antibody conjugations. The isotype controls have the function to define the unspecific fluorescence (negative cells) and the fluorescent regions (positive cells). In this study was selected monoclonal antibody (mAb) against canine erythrocyte antigen, produced in the Monoclonal Antibodies Laboratory - Blood Center of Botucatu, which reacts positively with dog red blood cells, but never with human leukocytes, having therefore, utility potential as negative control in flow cytometry. The purification mAb of IgG1 subclass was made by affinity chromatography in Sepharose Protein-A and the purification control was performed by electrophoresis in ágarose and polyacrylamide gels (SDS-PAGE). The purified immunoglobulin was conjugated to FITC and after was filtered in Sephadex G25 column to separation of labeled and unlabeled proteins. The conjugated mAb was tested against dog red blood cells and the conjugation success was verified by fluorescence tests, being the median positivity of 94.70. To the human leucocytes the positivity median was 0.03 against 0.50 of the commercial reagents. The nonparametric statistical tests of Wilcoxon and the correlation Spearman showed the efficiency and validate the isotype control produced in relation to the tested commercial reagents.
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Fluorescein-5-isothiocyanate , Blood , Immunoglobulin Isotypes , Immunoglobulin G , Immunoglobulins , Isothiocyanates , Electrophoresis , Electrophoresis, Polyacrylamide Gel , Hemotherapy Service , Flow Cytometry , Indicators and Reagents , Antibodies , Antibodies, Monoclonal , AntigensABSTRACT
OBJECTIVE: In this study, we evaluated whether the hypo-osmotic swelling test (HOST) can be used as a vital marker in combination with peanut agglutinin (PNA) - labeling in fresh and cryopreserved spermatozoa. MATERIALS AND METHODS: Human sperm populations were exposed to a hypo-osmotic medium for 60 minutes, and then incubated in a 1 µg/mL solution of the fluorescent dye Hoescht 33258 (H33258) for 10 minutes. Excess stain was removed by washing in phosphate-buffered saline (PBS) solution, and the pellet was resuspended in 100 µL of culture medium. Twenty microliters of this solution were subsequently smeared on a microscope slide, and fixed in ice-cold methanol to permeabilize the sperm membranes. The fixed smears were finally incubated in a 40-µg/mL FITC-PNA solution for 20 minutes. Simultaneous assessment of acrosome and viability scores was done in a fluorescent microscope equipped with appropriate filters and phase contrast illumination. The same slide was examined for FITC-PNA labeling, tail swelling, and for Hoechst-33258 staining by interchanging the filters and phase contrast optics. RESULTS: In fresh specimens, HOST was found to provide viability assessments comparable to those obtained using the H33258 method (r = 0.95). However, the results of HOST and H33258 were not correlated in cryopreserved specimens (r = 0.22). There was no alteration of PNA-labeling due to the HOST or H33258. CONCLUSIONS: FITC-PNA labeling in conjunction with the visualization of the morphological change induced by exposure to hypo-osmotic solution provides a simple but effective method for establishing the state of acrosomal membrane and viability in fresh human spermatozoa, but this technique is not reliable for cryopreserved ones.
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Adult , Humans , Male , Acrosome Reaction/physiology , Cryopreservation , Sperm Motility , Semen Preservation/methods , Spermatozoa/physiology , Cell Culture Techniques , Fluorescent Dyes , Osmolar Concentration , Peanut AgglutininABSTRACT
Two sources of adult stem cells that have aroused great interest are human bone marrow-derived mesenchymal stem cells (hMSCs) and human umbilical cord blood cells. hMSCs have been reported to maintain their ability to differentiate into neuronal lineage cells in the central nervous system. Therefore, transplantation of hMSCs represents an attractive new form of cellular therapy for clinical application in spinal cord injury (SCI). The aim of this study was to investigate how transplanted hMSCs from the venous circulation moved into a target zone of compression injury in the spinal cord of rats, and if they ameliorated the behavioral impairments associated with SCI. SCI in rats was induced by compressing the spinal cord for 30 s with an aneurysm clip. hMSCs labeled with cholera toxin subunit B conjugated to fluorescein isothiocyanate (CTX B-FITC) were injected intravenously through the tail vein or directly on the SCI site using a 27-g needle. Suspensions of hMSCs collected from adult humans were delivered at concentrations (1x10(6)cells/200 microliter) in 1 or 5 d after experimental SCI. After transplantation of hMSCs, the SCI regions displayed some endogenous background fluorescence, but CTX B-FITC-labeled hMSCs were clearly identifiable. They were observed in injured but not in intact areas; they were usually round or slightly elongated with a prominent nucleus. Only a few hMSCs were found in the spinal cord in each case but there were more cells in the rats injected at day one than at day five. This study confirmed that these were indeed transplanted hMSCs using antisera recognizing human-specific nuclei or human-specific mitochondria. Double immunofluorescence analysis showed the production of some neuronal and glial cell markers in the SCI lesions. Behavioral test scores of SCI rats treated with hMSCs at day one were significantly better than those for rats treated at day five and for the untreated SCI group. Thus, hMSCs appear to be beneficial in reversing the behavioral effects of SCI in this rat model, even when infused one day after injury. They might be a viable source of stem cells for the treatment of human neurological disorders.
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Adult , Animals , Humans , Rats , Adult Stem Cells , Aneurysm , Central Nervous System , Cholera Toxin , Fetal Blood , Fluorescein , Fluorescence , Fluorescent Antibody Technique , Immune Sera , Mesenchymal Stem Cells , Mitochondria , Models, Animal , Needles , Nervous System Diseases , Neuroglia , Neurons , Spinal Cord Injuries , Spinal Cord , Stem Cells , Suspensions , VeinsABSTRACT
Objective To prepare a novel annexin V and conjugate it with fluorescein isothiocyanate and test their binding ability to phosphatidylserine(PS)exposed erythrocytes.Methods The annexin V fused with an metal chelating binding site was obtained from pichia pastoris culturing and methanol induction expression.The annexin V was purified from the culture supernatant crude product by ion-exchange chromatography and ammonium sulfate precipitation.The annexin V was conjugated with fluorescein isothiocyanate in boric buffer and the conjugate annexin V-FITC was purified by ion-exchange chromatography.Sheep red blood cells were treated with Glutaraldehyde to expose membrane PS.The annexin V-FITC binding to PS exposed erythrocytes was investigated by fluorescent microscopy.Results The novel annexin V was purified and concentrated from pichia pastoris culture by ion-exchange chromatography and ammonium sulfate precipitation.After conjugating with FITC,the annexin V was found to bind PS exposed erythrocytes by fluorescent microscopy.Conclusion The annexin V expressed recombinantly in pichia pastoris retains the binding ability to PS exposed erythrocytes and is applicable to apoptosis detection in vitro.