-C1 double transfected cells. (DOCX 1342 kb) Further file five: Preparation of cartridges — различия между версиями

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Molecular Neurodegeneration (2016) 11:Web page 19 ofAbbreviations cCasp3: cleaved Casp3; CGCs: cerebellar granule cells; DsRed1: wild-type Discosoma red fluorescent protein; ECFP: enhanced cyan fluorescent protein; E-FRET: sensitized acceptor fluorescence; EYFP: enhanced yellow fluorescent protein; FLIM-FRET: donor fluorescence lifetime; FRET: fluorescence resonance [http://www.medchemexpress.com/Dalfopristin.html RP54476 manufacturer] energy transfer; FRPs: fluorescent reporter proteins; hCMV: human cytomegalovirus; HcRed1: humanized DsRed1; HPT: hours post-transfection; IAPs: inhibitors of apoptosis proteins; ICC: immunocytochemistry; LSCFM: laser scanning confocal fluorescence microscopy; NA: numerical aperture; NMDA: N-methyl-D-aspartate; NOND: naturally occurring neuronal death; OCCs: organotypic cerebellar cultures; p: plasmid; PCD: programmed cell death; RGCs: retinal ganglion cells; RNAi: RNA interference; ROI: region of interest; sh: quick hairpin; TCD: transfected cell density. Neuronal cell death: an overview of its different types in central and peripheral neurons. In: Lossi L, Merighi A, editors. Neuronal Cell Death. New York: Springer; 2015. p. 1?8. two. Hamburger V, Oppenheim RW. Naturally occurring neuronal death in vertebrates: neuroembryology. Boston: Birkh ser; 1990. p. 126?2. 3. We subjected OCCs to pharmacological remedies (see Table 1) and monitored them at diverse time intervals as much as twenty-four hours. At every single monitoring point, unique sets of digital pictures [https://dx.doi.org/10.1089/jir.2014.0001 title= jir.2014.0001] had been acquired according to the experimental paradigm employed (two pictures for single transfection experiments, 3 for doubleLossi et al. Molecular Neurodegeneration (2016) 11:Web page 19 ofAbbreviations cCasp3: cleaved Casp3; CGCs: cerebellar granule cells; DsRed1: wild-type Discosoma red fluorescent protein; ECFP: enhanced cyan fluorescent protein; E-FRET: sensitized acceptor fluorescence; EYFP: enhanced yellow fluorescent protein; FLIM-FRET: donor fluorescence lifetime; FRET: fluorescence resonance energy transfer; FRPs: fluorescent reporter proteins; hCMV: human cytomegalovirus; HcRed1: humanized DsRed1; HPT: hours post-transfection; IAPs: inhibitors of apoptosis proteins; ICC: immunocytochemistry; LSCFM: laser scanning confocal fluorescence microscopy; NA: numerical aperture; NMDA: N-methyl-D-aspartate; NOND: naturally occurring neuronal death; OCCs: organotypic cerebellar cultures; p: plasmid; PCD: programmed cell death; RGCs: retinal ganglion cells; RNAi: RNA interference; ROI: region of interest; sh: short hairpin; TCD: transfected cell density. Competing interests The authors declare that they have no competing interests. Authors' contributions LL conceived the study, participated in its design and coordination, performed ICC, wide-field microscopy studies and TCD analysis, helped to draft the manuscript. CC [https://dx.doi.org/10.3389/fnins.2013.00232 title= fnins.2013.00232] performed LSCFM, FRET measurements on fixed tissues, helped to prepare OCCs, and to execute ICC and statistics. SA performed LSCFM, FRET measurements on fixed and live tissues, and initial statistical evaluation. AM conceived the study, participated in its design and style and coordination, performed RNAi and inhibitor experiments, critically revised statistical evaluation, and drafted the manuscript. All authors have been involved in drafting the manuscript or revising it critically for essential intellectual content, read and authorized the final manuscript. Acknowledgements We're drastically indebted to Prof. Masayuki Miura for kindly offering us with the SCAT3-DEVD and SCAT3-DEVG probes and for useful comments and ideas about this work. We also wish to thank Dr. Rachel Altura for her type present of the HcRed1-C1-survivin plasmid. This function was supported by local grants in the University of Turin. Received: 18 August 2015 Accepted: 22 AprilReferences 1. Lossi L, Castagna C, Merighi A. Neuronal cell death: an overview of its various types in central and peripheral neurons. In: Lossi L, Merighi A, editors. Neuronal Cell Death. New York: Springer; 2015. p.
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Competing interests The authors [http://05961.net/comment/html/?308870.html 75, E934 941. 32. Picinato, M.C.; Haber, E.P.; Carpinelli, A.R.; Cipolla-Neto] declare that they've no competing interests. 1995;7:211?. four. Yuan J, Yankner BA. Apoptosis inside the nervous method. Nature. 2000;407:802?. 5. Thornberry NA, Lazebnik Y. Caspases: enemies inside. Science. 1998;281:1312?. 6. Cryns V, Yuan J. Proteases to die for. Ge.-C1 double transfected cells. (DOCX 1342 kb) More file five: Preparation of cartridges for Casp3 RNAi experiments. Protocol to prepare many plasmid DNA-coated gold particles for biolistic experiments. (DOCX 18 kb)To measure FRET in living OCCs, 4 DIV cultures transfected with probe-containing plasmids were transferred in to the incubation chamber mounted around the SP5 stage. We subjected OCCs to pharmacological treatments (see Table 1) and monitored them at unique time intervals up to twenty-four hours. At each and every monitoring point, various sets of digital pictures [https://dx.doi.org/10.1089/jir.2014.0001 title= jir.2014.0001] were acquired in accordance with the experimental paradigm employed (two photos for single transfection experiments, 3 for doubleLossi et al. Molecular Neurodegeneration (2016) 11:Web page 19 ofAbbreviations cCasp3: cleaved Casp3; CGCs: cerebellar granule cells; DsRed1: wild-type Discosoma red fluorescent protein; ECFP: enhanced cyan fluorescent protein; E-FRET: sensitized acceptor fluorescence; EYFP: enhanced yellow fluorescent protein; FLIM-FRET: donor fluorescence lifetime; FRET: fluorescence resonance power transfer; FRPs: fluorescent reporter proteins; hCMV: human cytomegalovirus; HcRed1: humanized DsRed1; HPT: hours post-transfection; IAPs: inhibitors of apoptosis proteins; ICC: immunocytochemistry; LSCFM: laser scanning confocal fluorescence microscopy; NA: numerical aperture; NMDA: N-methyl-D-aspartate; NOND: naturally occurring neuronal death; OCCs: organotypic cerebellar cultures; p: plasmid; PCD: programmed cell death; RGCs: retinal ganglion cells; RNAi: RNA interference; ROI: region of interest; sh: quick hairpin; TCD: transfected cell density. Competing interests The authors declare that they've no competing interests. Authors' contributions LL conceived the study, participated in its design and coordination, performed ICC, wide-field microscopy studies and TCD analysis, helped to draft the manuscript. CC [https://dx.doi.org/10.3389/fnins.2013.00232 title= fnins.2013.00232] performed LSCFM, FRET measurements on fixed tissues, helped to prepare OCCs, and to carry out ICC and statistics. SA performed LSCFM, FRET measurements on fixed and reside tissues, and initial statistical analysis. AM conceived the study, participated in its design and coordination, performed RNAi and inhibitor experiments, critically revised statistical analysis, and drafted the manuscript. All authors have already been involved in drafting the manuscript or revising it critically for critical intellectual content, read and authorized the final manuscript. Acknowledgements We're significantly indebted to Prof. Masayuki Miura for kindly offering us with the SCAT3-DEVD and SCAT3-DEVG probes and for valuable comments and recommendations about this operate. We also want to thank Dr. Rachel Altura for her sort present with the HcRed1-C1-survivin plasmid. This operate was supported by local grants in the University of Turin. Received: 18 August 2015 Accepted: 22 AprilReferences 1. Lossi L, Castagna C, Merighi A. Neuronal cell death: an overview of its diverse types in central and peripheral neurons. In: Lossi L, Merighi A, editors. Neuronal Cell Death. New York: Springer; 2015. p. 1?eight. two. Hamburger V, Oppenheim RW. Naturally occurring neuronal death in vertebrates: neuroembryology. Boston: Birkh ser; 1990. p. 126?two. 3. Yuan J. Molecular handle of life and death. Curr Opin Cell Biol. 1995;7:211?. 4. Yuan J, Yankner BA.

Текущая версия на 05:30, 10 февраля 2018

Competing interests The authors 75, E934 941. 32. Picinato, M.C.; Haber, E.P.; Carpinelli, A.R.; Cipolla-Neto declare that they've no competing interests. 1995;7:211?. four. Yuan J, Yankner BA. Apoptosis inside the nervous method. Nature. 2000;407:802?. 5. Thornberry NA, Lazebnik Y. Caspases: enemies inside. Science. 1998;281:1312?. 6. Cryns V, Yuan J. Proteases to die for. Ge.-C1 double transfected cells. (DOCX 1342 kb) More file five: Preparation of cartridges for Casp3 RNAi experiments. Protocol to prepare many plasmid DNA-coated gold particles for biolistic experiments. (DOCX 18 kb)To measure FRET in living OCCs, 4 DIV cultures transfected with probe-containing plasmids were transferred in to the incubation chamber mounted around the SP5 stage. We subjected OCCs to pharmacological treatments (see Table 1) and monitored them at unique time intervals up to twenty-four hours. At each and every monitoring point, various sets of digital pictures title= jir.2014.0001 were acquired in accordance with the experimental paradigm employed (two photos for single transfection experiments, 3 for doubleLossi et al. Molecular Neurodegeneration (2016) 11:Web page 19 ofAbbreviations cCasp3: cleaved Casp3; CGCs: cerebellar granule cells; DsRed1: wild-type Discosoma red fluorescent protein; ECFP: enhanced cyan fluorescent protein; E-FRET: sensitized acceptor fluorescence; EYFP: enhanced yellow fluorescent protein; FLIM-FRET: donor fluorescence lifetime; FRET: fluorescence resonance power transfer; FRPs: fluorescent reporter proteins; hCMV: human cytomegalovirus; HcRed1: humanized DsRed1; HPT: hours post-transfection; IAPs: inhibitors of apoptosis proteins; ICC: immunocytochemistry; LSCFM: laser scanning confocal fluorescence microscopy; NA: numerical aperture; NMDA: N-methyl-D-aspartate; NOND: naturally occurring neuronal death; OCCs: organotypic cerebellar cultures; p: plasmid; PCD: programmed cell death; RGCs: retinal ganglion cells; RNAi: RNA interference; ROI: region of interest; sh: quick hairpin; TCD: transfected cell density. Competing interests The authors declare that they've no competing interests. Authors' contributions LL conceived the study, participated in its design and coordination, performed ICC, wide-field microscopy studies and TCD analysis, helped to draft the manuscript. CC title= fnins.2013.00232 performed LSCFM, FRET measurements on fixed tissues, helped to prepare OCCs, and to carry out ICC and statistics. SA performed LSCFM, FRET measurements on fixed and reside tissues, and initial statistical analysis. AM conceived the study, participated in its design and coordination, performed RNAi and inhibitor experiments, critically revised statistical analysis, and drafted the manuscript. All authors have already been involved in drafting the manuscript or revising it critically for critical intellectual content, read and authorized the final manuscript. Acknowledgements We're significantly indebted to Prof. Masayuki Miura for kindly offering us with the SCAT3-DEVD and SCAT3-DEVG probes and for valuable comments and recommendations about this operate. We also want to thank Dr. Rachel Altura for her sort present with the HcRed1-C1-survivin plasmid. This operate was supported by local grants in the University of Turin. Received: 18 August 2015 Accepted: 22 AprilReferences 1. Lossi L, Castagna C, Merighi A. Neuronal cell death: an overview of its diverse types in central and peripheral neurons. In: Lossi L, Merighi A, editors. Neuronal Cell Death. New York: Springer; 2015. p. 1?eight. two. Hamburger V, Oppenheim RW. Naturally occurring neuronal death in vertebrates: neuroembryology. Boston: Birkh ser; 1990. p. 126?two. 3. Yuan J. Molecular handle of life and death. Curr Opin Cell Biol. 1995;7:211?. 4. Yuan J, Yankner BA.