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Robert Webb - One of the best experts on this subject based on the ideXlab platform.
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Differential Staining combined with TUNEL labelling to detect apoptosis in preimplantation
2005Co-Authors: Ali A. Fouladi-nashta, Ramiro Alberio, Mojtaba Kafi, B Nicholas, K H S Campbell, Robert WebbAbstract:Development of accurate laboratory methods to assess embryo quality will improve the efficiency of embryo production from in-vitro culture systems. Currently, the techniques of TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end (TUNEL) labelling for the detection of apoptosis, and Differential Staining for determining the ratio of inner cell mass (ICM) to trophectoderm (TE) cells, are used separately to assess embryo quality in a range of different species. This paper reports a unique, simple and fast method for the assessment of embryo quality using Differential Staining of TE and ICM, but combined with TUNEL labelling (DST Staining). This technique was used to investigate the effect of serum supplementation on total cell number, ICM:TE ratio and apoptosis index after in-vitro production of bovine embryos. Serum supplementation increased total cell number (P < 0.01), but reduced the ratio of ICM:TE cells. No differences were observed in the number of apoptotic nuclei between treatments, or in the localization of the apoptotic nuclei. However, more apoptotic nuclei were observed in ICM than TE cells in both culture groups. In conclusion, using DST, it has been possible to carry out both a qualitative and quantitative analysis of embryos produced using the two different methods. DST provides a means of assessing the effect of culture conditions on cell number of both embryo compartments (ICM and TE), as well as providing information on the localization of apoptotic nuclei within the blastocyst.
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Differential Staining combined with TUNEL labelling to detect apoptosis in preimplantation bovine embryos
Reproductive biomedicine online, 2005Co-Authors: Ali A. Fouladi-nashta, Ramiro Alberio, Mojtaba Kafi, B Nicholas, K H S Campbell, Robert WebbAbstract:Development of accurate laboratory methods to assess embryo quality will improve the efficiency of embryo production from in-vitro culture systems. Currently, the techniques of TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end (TUNEL) labelling for the detection of apoptosis, and Differential Staining for determining the ratio of inner cell mass (ICM) to trophectoderm (TE) cells, are used separately to assess embryo quality in a range of different species. This paper reports a unique, simple and fast method for the assessment of embryo quality using Differential Staining of TE and ICM, but combined with TUNEL labelling (DST Staining). This technique was used to investigate the effect of serum supplementation on total cell number, ICM:TE ratio and apoptosis index after in-vitro production of bovine embryos. Serum supplementation increased total cell number (P < 0.01), but reduced the ratio of ICM:TE cells. No differences were observed in the number of apoptotic nuclei between treatments, or in the localization of the apoptotic nuclei. However, more apoptotic nuclei were observed in ICM than TE cells in both culture groups. In conclusion, using DST, it has been possible to carry out both a qualitative and quantitative analysis of embryos produced using the two different methods. DST provides a means of assessing the effect of culture conditions on cell number of both embryo compartments (ICM and TE), as well as providing information on the localization of apoptotic nuclei within the blastocyst.
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151 A SIMPLE AND FAST METHOD FOR CONCURRENT Differential Staining AND TUNEL LABELLING OF BOVINE BLASTOCYSTS
Reproduction Fertility and Development, 2004Co-Authors: Ali A. Fouladi-nashta, Ramiro Alberio, B Nicholas, K H S Campbell, Robert WebbAbstract:Currently techniques of TUNEL labelling for detection of apoptosis and Differential Staining for counting the ratio of inner cell mass (ICM) to trophoectoderm (TE) cells are used separately for assessment of embryo quality in different species. The majority of these techniques are antibody-based, and time-consuming, frequently giving inconsistent results. Here we report on the development of a simple and fast method for simultaneous Differential Staining and TUNEL labelling of bovine embryos. Cleaved embryos produced from in vitro-matured and fertilized oocytes were cultured to the blastocyst stage in synthetic oviductal fluid culture medium (SOF) supplemented with 4 mg mL−1 BSA and 5% FCS. Embryos were partially permeabilized in 0.5% Triton X-100 solution containing 2 μM mL−1 TOTO-3 dye (Molecular Probes, Eugene, OR, USA) for 30 s. TOTO-3 is a cell-impermeant nucleic acid dye; thus only permeabilized cells are stained red. The embryos were then quickly washed in PBS containing 3 mg mL−1 PVA, fixed for 15 min at RT in 4% paraformaldehyde containing 10 μg mL−1 Hoescht, and TUNEL-labelled using a Cell Death Kit (Roche Applied Science, East Sussex, UI) for 30 min at 37°C in a humid chamber. The embryos were then treated with RNase A (50 U mL−1) for 30 min at 37°C, washed and mounted in a small drop of glycerol on a glass slide. RQ1-DNase (3 U mL−1)-treated embryos were used as a positive control. After three-dimensional reconstruction using a Leica TCS SP2 confocal microscope, we determined the number of ICM (blue), TE (red) and apoptotic nuclei (green). Only peripheral cells of the blastocysts were labelled red, indicating that TE cells were permeabilized by the short exposure to the detergent Triton. ICM cells were consistently stained blue by the cell permeant dye Hoechst. Apoptotic nuclei were found in both types of cells. More consistent Differential Staining was observed in hatched blastocysts (n = 30) than in zona-enclosed blastocysts (n = 35); also, more apoptotic nuclei were observed. No differences were found in the consistency of the technique for embryos grown with or without FCS. When compared to dual Staining without Tunel, no differences in cell number (74 ± 22) , and ICM/TE ratio (0.28 ± 0.06) were detected, indicating that the Tunel procedure does not affect the labeling of the DNA. Preliminary observations also indicate that this method can be successfully applied to porcine and ovine embryos. This technique has the advantage of being fast and can be applied for assessment of embryo quality. It can also be used to determine the time and origin of ICM and TE differentiation while monitoring the degree of apoptosis in different culture systems and in different species. This work was in part supported by Department of Environment Food and Rural Affairs (Defra) UK.
Ali A. Fouladi-nashta - One of the best experts on this subject based on the ideXlab platform.
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Differential Staining combined with TUNEL labelling to detect apoptosis in preimplantation
2005Co-Authors: Ali A. Fouladi-nashta, Ramiro Alberio, Mojtaba Kafi, B Nicholas, K H S Campbell, Robert WebbAbstract:Development of accurate laboratory methods to assess embryo quality will improve the efficiency of embryo production from in-vitro culture systems. Currently, the techniques of TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end (TUNEL) labelling for the detection of apoptosis, and Differential Staining for determining the ratio of inner cell mass (ICM) to trophectoderm (TE) cells, are used separately to assess embryo quality in a range of different species. This paper reports a unique, simple and fast method for the assessment of embryo quality using Differential Staining of TE and ICM, but combined with TUNEL labelling (DST Staining). This technique was used to investigate the effect of serum supplementation on total cell number, ICM:TE ratio and apoptosis index after in-vitro production of bovine embryos. Serum supplementation increased total cell number (P < 0.01), but reduced the ratio of ICM:TE cells. No differences were observed in the number of apoptotic nuclei between treatments, or in the localization of the apoptotic nuclei. However, more apoptotic nuclei were observed in ICM than TE cells in both culture groups. In conclusion, using DST, it has been possible to carry out both a qualitative and quantitative analysis of embryos produced using the two different methods. DST provides a means of assessing the effect of culture conditions on cell number of both embryo compartments (ICM and TE), as well as providing information on the localization of apoptotic nuclei within the blastocyst.
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Differential Staining combined with TUNEL labelling to detect apoptosis in preimplantation bovine embryos
Reproductive biomedicine online, 2005Co-Authors: Ali A. Fouladi-nashta, Ramiro Alberio, Mojtaba Kafi, B Nicholas, K H S Campbell, Robert WebbAbstract:Development of accurate laboratory methods to assess embryo quality will improve the efficiency of embryo production from in-vitro culture systems. Currently, the techniques of TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end (TUNEL) labelling for the detection of apoptosis, and Differential Staining for determining the ratio of inner cell mass (ICM) to trophectoderm (TE) cells, are used separately to assess embryo quality in a range of different species. This paper reports a unique, simple and fast method for the assessment of embryo quality using Differential Staining of TE and ICM, but combined with TUNEL labelling (DST Staining). This technique was used to investigate the effect of serum supplementation on total cell number, ICM:TE ratio and apoptosis index after in-vitro production of bovine embryos. Serum supplementation increased total cell number (P < 0.01), but reduced the ratio of ICM:TE cells. No differences were observed in the number of apoptotic nuclei between treatments, or in the localization of the apoptotic nuclei. However, more apoptotic nuclei were observed in ICM than TE cells in both culture groups. In conclusion, using DST, it has been possible to carry out both a qualitative and quantitative analysis of embryos produced using the two different methods. DST provides a means of assessing the effect of culture conditions on cell number of both embryo compartments (ICM and TE), as well as providing information on the localization of apoptotic nuclei within the blastocyst.
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151 A SIMPLE AND FAST METHOD FOR CONCURRENT Differential Staining AND TUNEL LABELLING OF BOVINE BLASTOCYSTS
Reproduction Fertility and Development, 2004Co-Authors: Ali A. Fouladi-nashta, Ramiro Alberio, B Nicholas, K H S Campbell, Robert WebbAbstract:Currently techniques of TUNEL labelling for detection of apoptosis and Differential Staining for counting the ratio of inner cell mass (ICM) to trophoectoderm (TE) cells are used separately for assessment of embryo quality in different species. The majority of these techniques are antibody-based, and time-consuming, frequently giving inconsistent results. Here we report on the development of a simple and fast method for simultaneous Differential Staining and TUNEL labelling of bovine embryos. Cleaved embryos produced from in vitro-matured and fertilized oocytes were cultured to the blastocyst stage in synthetic oviductal fluid culture medium (SOF) supplemented with 4 mg mL−1 BSA and 5% FCS. Embryos were partially permeabilized in 0.5% Triton X-100 solution containing 2 μM mL−1 TOTO-3 dye (Molecular Probes, Eugene, OR, USA) for 30 s. TOTO-3 is a cell-impermeant nucleic acid dye; thus only permeabilized cells are stained red. The embryos were then quickly washed in PBS containing 3 mg mL−1 PVA, fixed for 15 min at RT in 4% paraformaldehyde containing 10 μg mL−1 Hoescht, and TUNEL-labelled using a Cell Death Kit (Roche Applied Science, East Sussex, UI) for 30 min at 37°C in a humid chamber. The embryos were then treated with RNase A (50 U mL−1) for 30 min at 37°C, washed and mounted in a small drop of glycerol on a glass slide. RQ1-DNase (3 U mL−1)-treated embryos were used as a positive control. After three-dimensional reconstruction using a Leica TCS SP2 confocal microscope, we determined the number of ICM (blue), TE (red) and apoptotic nuclei (green). Only peripheral cells of the blastocysts were labelled red, indicating that TE cells were permeabilized by the short exposure to the detergent Triton. ICM cells were consistently stained blue by the cell permeant dye Hoechst. Apoptotic nuclei were found in both types of cells. More consistent Differential Staining was observed in hatched blastocysts (n = 30) than in zona-enclosed blastocysts (n = 35); also, more apoptotic nuclei were observed. No differences were found in the consistency of the technique for embryos grown with or without FCS. When compared to dual Staining without Tunel, no differences in cell number (74 ± 22) , and ICM/TE ratio (0.28 ± 0.06) were detected, indicating that the Tunel procedure does not affect the labeling of the DNA. Preliminary observations also indicate that this method can be successfully applied to porcine and ovine embryos. This technique has the advantage of being fast and can be applied for assessment of embryo quality. It can also be used to determine the time and origin of ICM and TE differentiation while monitoring the degree of apoptosis in different culture systems and in different species. This work was in part supported by Department of Environment Food and Rural Affairs (Defra) UK.
P G Cionini - One of the best experts on this subject based on the ideXlab platform.
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the chromosome complement of olea europaea l characterization by Differential Staining of the chromatin and in situ hybridization of highly repeated dna sequences
Chromosome Research, 2000Co-Authors: S Minelli, F Maggini, M T Gelati, A Angiolillo, P G CioniniAbstract:The chromosome complement of olive (Olea europaea L.) has been characterized by Differential Staining of the chromatin and chromosomal localization of highly repeated DNA sequences and ribosomal cistrons. DAPI Staining produces different-sized positive bands in various locations on all the chromosomes. By combining this band pattern with the results obtained from cytological hybridization of OeTaq80, OeTaq178, and OeGEM86 DNA tandem repeats, most of the pairs can be distinguished from each other, in spite of the large number of chromosomes (2n=46), their small size and similar morphology. Different tandem-repeated DNA sequences may be contained into single heterochromatic chromosome regions, even though there are regions where repeats of only one family are present. OeTaq80- and OeGEM86-related DNA sequences are rather specific to the heterochromatin at the chromosome ends, while most sequences related to the longer OeTaq178 probe are confined to interstitial heterochromatin. Some exceptions suggest that major chromosomal rearrangements occurred during genome evolution. Polymorphism, which may differentiate olive cultivars, was observed within chromosome pairs I, V, and VII.
Chen Xianjun - One of the best experts on this subject based on the ideXlab platform.
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Study on the related factors for the Differential Staining results of sister chromatid
Journal of Shanxi Medical University, 2012Co-Authors: Chen XianjunAbstract:Objective To explore the effects of frozen storage time of bromodeoxyuridine(BrdU) solution,storage time of chromosome slides,ultraviolet exposure time and temperature on the Differential Staining results of sister chromatid,and to figure out whether it is feasible to put the technique as a classroom experiment project.Methods The chromosome slides were prepared by the standard methods of peripheral blood lymphocyte culture and chromosome preparation.The cells were cultured with the BrdU solutions prepared freshly and stored for 10 years at-20 ℃,respectively.The chromosome slides prepared freshly and stored at-20 ℃ for 1,2,3,4,5 weeks were taken for the ultraviolet irradiation at 50,56 and 60 ℃ for 10,20 and 30 min,respectively,and then stained with Giemsa for 10 min at the room temperature.The effects of the factors on the Differential Staining were evaluated according to the Staining results and colour difference of the chromatids.Results The Staining results of chromatids showed no significant difference after incorporating into DNA with BrdU prepared freshly and stored at-20 ℃ for 10 years,and the most appropriate final concentration of BrdU was 10 μg/ml.Good Differential Staining results were obtained from the chromosome slides prepared freshly and stored at-20 ℃ for 1-5 weeks by UV radiation for 10-30 min.Conclusion BrdU solution can be stored at-20 ℃ for a long-term without affecting the chemical incorporation into DNA.Chromosome slides stored at-20 ℃ can be used for sister chromatid Differential Staining in 5 weeks after preparation.Sister chromatid Differential Staining is a technique which is tolerant toward the changes of the related factors,and it is entirely feasible as a classroom experiment after the factors are improved.
Ramiro Alberio - One of the best experts on this subject based on the ideXlab platform.
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Differential Staining combined with TUNEL labelling to detect apoptosis in preimplantation
2005Co-Authors: Ali A. Fouladi-nashta, Ramiro Alberio, Mojtaba Kafi, B Nicholas, K H S Campbell, Robert WebbAbstract:Development of accurate laboratory methods to assess embryo quality will improve the efficiency of embryo production from in-vitro culture systems. Currently, the techniques of TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end (TUNEL) labelling for the detection of apoptosis, and Differential Staining for determining the ratio of inner cell mass (ICM) to trophectoderm (TE) cells, are used separately to assess embryo quality in a range of different species. This paper reports a unique, simple and fast method for the assessment of embryo quality using Differential Staining of TE and ICM, but combined with TUNEL labelling (DST Staining). This technique was used to investigate the effect of serum supplementation on total cell number, ICM:TE ratio and apoptosis index after in-vitro production of bovine embryos. Serum supplementation increased total cell number (P < 0.01), but reduced the ratio of ICM:TE cells. No differences were observed in the number of apoptotic nuclei between treatments, or in the localization of the apoptotic nuclei. However, more apoptotic nuclei were observed in ICM than TE cells in both culture groups. In conclusion, using DST, it has been possible to carry out both a qualitative and quantitative analysis of embryos produced using the two different methods. DST provides a means of assessing the effect of culture conditions on cell number of both embryo compartments (ICM and TE), as well as providing information on the localization of apoptotic nuclei within the blastocyst.
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Differential Staining combined with TUNEL labelling to detect apoptosis in preimplantation bovine embryos
Reproductive biomedicine online, 2005Co-Authors: Ali A. Fouladi-nashta, Ramiro Alberio, Mojtaba Kafi, B Nicholas, K H S Campbell, Robert WebbAbstract:Development of accurate laboratory methods to assess embryo quality will improve the efficiency of embryo production from in-vitro culture systems. Currently, the techniques of TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end (TUNEL) labelling for the detection of apoptosis, and Differential Staining for determining the ratio of inner cell mass (ICM) to trophectoderm (TE) cells, are used separately to assess embryo quality in a range of different species. This paper reports a unique, simple and fast method for the assessment of embryo quality using Differential Staining of TE and ICM, but combined with TUNEL labelling (DST Staining). This technique was used to investigate the effect of serum supplementation on total cell number, ICM:TE ratio and apoptosis index after in-vitro production of bovine embryos. Serum supplementation increased total cell number (P < 0.01), but reduced the ratio of ICM:TE cells. No differences were observed in the number of apoptotic nuclei between treatments, or in the localization of the apoptotic nuclei. However, more apoptotic nuclei were observed in ICM than TE cells in both culture groups. In conclusion, using DST, it has been possible to carry out both a qualitative and quantitative analysis of embryos produced using the two different methods. DST provides a means of assessing the effect of culture conditions on cell number of both embryo compartments (ICM and TE), as well as providing information on the localization of apoptotic nuclei within the blastocyst.
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151 A SIMPLE AND FAST METHOD FOR CONCURRENT Differential Staining AND TUNEL LABELLING OF BOVINE BLASTOCYSTS
Reproduction Fertility and Development, 2004Co-Authors: Ali A. Fouladi-nashta, Ramiro Alberio, B Nicholas, K H S Campbell, Robert WebbAbstract:Currently techniques of TUNEL labelling for detection of apoptosis and Differential Staining for counting the ratio of inner cell mass (ICM) to trophoectoderm (TE) cells are used separately for assessment of embryo quality in different species. The majority of these techniques are antibody-based, and time-consuming, frequently giving inconsistent results. Here we report on the development of a simple and fast method for simultaneous Differential Staining and TUNEL labelling of bovine embryos. Cleaved embryos produced from in vitro-matured and fertilized oocytes were cultured to the blastocyst stage in synthetic oviductal fluid culture medium (SOF) supplemented with 4 mg mL−1 BSA and 5% FCS. Embryos were partially permeabilized in 0.5% Triton X-100 solution containing 2 μM mL−1 TOTO-3 dye (Molecular Probes, Eugene, OR, USA) for 30 s. TOTO-3 is a cell-impermeant nucleic acid dye; thus only permeabilized cells are stained red. The embryos were then quickly washed in PBS containing 3 mg mL−1 PVA, fixed for 15 min at RT in 4% paraformaldehyde containing 10 μg mL−1 Hoescht, and TUNEL-labelled using a Cell Death Kit (Roche Applied Science, East Sussex, UI) for 30 min at 37°C in a humid chamber. The embryos were then treated with RNase A (50 U mL−1) for 30 min at 37°C, washed and mounted in a small drop of glycerol on a glass slide. RQ1-DNase (3 U mL−1)-treated embryos were used as a positive control. After three-dimensional reconstruction using a Leica TCS SP2 confocal microscope, we determined the number of ICM (blue), TE (red) and apoptotic nuclei (green). Only peripheral cells of the blastocysts were labelled red, indicating that TE cells were permeabilized by the short exposure to the detergent Triton. ICM cells were consistently stained blue by the cell permeant dye Hoechst. Apoptotic nuclei were found in both types of cells. More consistent Differential Staining was observed in hatched blastocysts (n = 30) than in zona-enclosed blastocysts (n = 35); also, more apoptotic nuclei were observed. No differences were found in the consistency of the technique for embryos grown with or without FCS. When compared to dual Staining without Tunel, no differences in cell number (74 ± 22) , and ICM/TE ratio (0.28 ± 0.06) were detected, indicating that the Tunel procedure does not affect the labeling of the DNA. Preliminary observations also indicate that this method can be successfully applied to porcine and ovine embryos. This technique has the advantage of being fast and can be applied for assessment of embryo quality. It can also be used to determine the time and origin of ICM and TE differentiation while monitoring the degree of apoptosis in different culture systems and in different species. This work was in part supported by Department of Environment Food and Rural Affairs (Defra) UK.