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