Isolering og kultur av nervus, Trochlear, og spinal motor neurons fra prenatal ISLMN: GFP transgene mus
Summary
Dette arbeidet presenterer en protokoll for å gi homogene cellekulturer av primære nervus, trochlear, og spinal motor neurons. Disse kulturene kan brukes til sammenlignende analyser av morfologiske, cellulære, molekylære, og elektrofysiologisk karakteristikker av øye og spinal motor neurons.
Abstract
Nervus neurons (CN3s) og trochlear neurons (CN4s) viser bemerkelsesverdig motstand mot degenerative motoriske Nevron sykdommer som amyotrofisk lateral sklerose (ALS) sammenlignet med spinal motor neurons (SMNs). Evnen til å isolere og kultur primær mus CN3s, CN4s og SMNs ville gi en tilnærming til studie mekanismer underliggende denne selektive sårbarheten. Til dags dato bruker de fleste protokollene heterogene cellekulturer, som kan forvirre tolkningen av eksperimentelle utfall. For å minimere problemene knyttet til blandet celle populasjoner, rene kulturer er uunnværlig. Her, den første protokollen beskriver i detalj hvordan du effektivt renser og dyrke CN3s/CN4s sammen SMNs kolleger fra samme embryo bruker embryonale dagen 11,5 (E 11.5) ISLMN: GFP transgene Mouse embryo. Protokollen gir detaljer om vevet disseksjon og dissosiasjon, FACS-baserte celle isolasjon, og in vitro dyrking av celler fra CN3/CN4 og SMN kjerner. Denne protokollen legger til en roman in vitro CN3/CN4 kultur system til eksisterende protokoller og samtidig gir en ren Art-og alder-matchet SMN kultur for sammenligning. Analyser som fokuserer på morfologiske, cellulære, molekylære og elektrofysiologisk karakteristikker av motor neurons er gjennomførbart i dette kultur systemet. Denne protokollen vil gjøre forskning i mekanismer som definerer motorisk nerve utvikling, selektiv sårbarhet, og sykdom.
Introduction
Kulturen i primær motor neurons er et kraftig verktøy som gjør det mulig å studere neuronal utvikling, funksjon, og mottakelighet for eksogene stressfaktorer. Motoriske Nevron kulturer er spesielt nyttig for studiet av nevrodegenerative sykdommer som amyotrofisk lateral sklerose (ALS)1,2, hvis sykdom mekanismer er ufullstendig forstått. Interessant, til tross for betydelig celle død spinal motor neurons (SMNs) i både ALS pasienter og ALS modell mus, celle død i nervus neurons (CN3s) og trochlear neurons (CN4s) er relativt knappe1,3,4,5,6,7,8,9. Derfor, komparative analyser av rene kulturer av CN3s/CN4s og SMNs kan gi viktige ledetråder om mekanismer underliggende relative sårbarhet. Dessverre har en stor barriere mot slike analyser vært manglende evne til å vokse renset kulturer av disse motor neurons.
Mange protokoller har blitt beskrevet for rensing av SMNs fra dyremodeller. De fleste av disse protokollene bruker tetthet gradientsentrifugering 10,11,12 og/eller p75ntR-antistoff-basert celle-sortering panorering teknikker13,14,15,16. Density gradient sentrifugering utnytter den større størrelsen på SMNs i forhold til andre spinal celler, mens p75ntR er et ekstracellulære protein uttrykt utelukkende av SMNs i ryggmargen. Nesten 100% ren SMN kulturer har blitt generert av en eller begge av disse protokollene11,12,14. Imidlertid har disse protokollene ikke lykkes i å generere CN3/CN4 kulturer fordi CN3s/CN4s ikke uttrykke p75ntR, og andre spesifikke CN3/CN4 markører har ikke blitt identifisert. De er også mindre enn SMNs, og derfor vanskeligere å isolere basert på størrelse. I stedet har in vitro studier av CN3s eller CN4s stolt på dissosiert17,18,19,20,21, explant17,22,23,24,25,26, og Slice27,28 kulturer, som er sammensatt av heterogene celletyper, og ingen protokoller har eksistert i isolasjon og kultur av primære CN3s eller CN4s.
Her er en protokoll beskrevet for visualisering, isolering, rensing, og dyrking av CN3s, CN4s, og SMNs fra samme embryonale dagen 11,5 (E 11.5) ISLMN: GFP transgene mus29 (figur 1, figur 2a). ISLMN: GFP spesielt etiketter motor NEURONS med en farnesylated GFP som lokaliseres til cellemembranen. Denne protokollen muliggjør arter-og alder-matchet sammenligning av flere typer motor neurons for å belyse patologiske mekanismer i motorisk Nevron sykdom.
Protocol
Representative Results
Discussion
Historisk, in vitro studier av CN3 og/eller CN4 motor neurons har stolt på heterogene kulturer som dissosiert17,18,19,20,21, explant17,22,23,24,25,26, og Slice<su…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
Vi takker Brigitte Pettmann (Biogen, Cambridge, MA, USA) for instruksjon i SMN disseksjon teknikker; The Dana Farber Cancer Institute Flow flowcytometri Facility, den immunologi Division Flow flowcytometri Facility av Harvard Medical School, The Joslin diabetes Center Flow flowcytometri Core, Brigham og Women ‘ s Hospital Flow flowcytometri Core, og Boston Children ‘ s Hospital Flow flowcytometri Research Facility for FACS isolering av primær motor neurons; A.A. Nugent, A.P. Tenney, A.S. Lee, E.H. Nguyen, M.F. Rose, flere Engle laboratorium medlemmer, og Project ALS konsortium medlemmer for teknisk assistanse og gjennomtenkt diskusjon. Denne studien ble støttet av Project ALS. I tillegg R.F. ble finansiert av Japan Heart Foundation/Bayer Yakuhin Research Grant utlandet og NIH Training stipend i genetikk T32 GM007748; JJ ble støttet av NIH/NEI treningsprogram i den molekylære baser av øyesykdommer (5T32EY007145-16) gjennom Schepens Eye Research Institute og av utviklingsmessige Nevrologi Training program postdoktor Fellowship (5T32NS007473-19) gjennom Boston Children ‘ s Hospital; M.s. W ble støttet av NEI (5K08EY027850) og Children ‘ s Hospital Oftalmologi Foundation (fakultet Discovery Award); og E.C.E. er en Howard Hughes Medical Institute etterforsker.
Materials
| Alexa Fluor 488-conjugated goat anti-mouse IgG (H+L) | Thermo Fisher Scientific | A-11001 | 1:400 |
| Alexa Fluor 594-conjugated F(ab')2 goat anti-rabbit IgG (H+L) | Thermo Fisher Scientific | A-11072 | 1:400 |
| B27 Supplement (50X), serum free | Thermo Fisher Scientific | 17504-044 | |
| BD FACSAria llu SORP Flow Cytometer | BD Bioscience | – | This has 4 laser system equipped with 405, 488, 594, and 640 nm lasers. |
| BD Falcon 70μm Nylon Cell Strainers | CORNING | 352350 | For filtering the dissociating cells before FACS. |
| BD Falcon Round Bottom Test Tubes With Snap Cap | CORNING | 352054 | |
| BDNF Human | ProSpec-Tany TechnoGene, Ltd. | CYT-207 | |
| Cell Culture microplate, 96 well, PS, F-bottom (Chimney Well) | Greiner Bio-One International | 655090 | We tried multiple 96-well dishes and this was the best one for culture and analyses after ICC |
| Circular Cover Glasses for microscopy | Karl Hecht & Assistent | 1001/14 | We used this coverslip since the area was large (diamater: 14 mm). |
| CNTF Human | ProSpec-Tany TechnoGene, Ltd. | CYT-272 | |
| Cyclopiazonic acid from Penicillium cyclopium | Sigma-Aldrich | C1530 | CPA. One of ER stressors. |
| 4′,6-diamidino-2-phenylinodole (DAPI) | Thermo Fisher Scientific | D1306 | |
| Dimethyl sulfoxide | Sigma-Aldrich | D2650 | DMSO |
| Dumont #5 Forceps Inox Tip Size .05 x .01 mm Biologie Tips | Roboz Surgical Instrument | RS-5015 | |
| Forskolin | Thermo Fisher Scientific | BP25205 | |
| GDNF Human | ProSpec-Tany TechnoGene, Ltd. | CYT-305 | |
| GlutaMAX supplement | Thermo Fisher Scientific | 35050-061 | |
| Hanks’ Balanced Salt Solution (HBSS) | Thermo Fisher Scientific | 14175-095 | |
| Hibernate E | BrainBits | HE | |
| Hibernate E low fluorescence | BrainBits | HELF | Fluorescence which hinders observation of embryo's GFP expressions should be low. |
| Horse serum, heat inactivated, New Zealand origin | Thermo Fisher Scientific | 26050-070 | |
| IBMX | Tocris Cookson | 2845 | Isobutylmethylxanthine |
| Laminin | Thermo Fisher Scientific | 23017-015 | |
| Leibovitz’s L15 medium | Thermo Fisher Scientific | 11415064 | |
| 2-Mercaptoethanol | Sigma-Aldrich | M6250 | |
| Micro Dissecting Scissors | Roboz Surgical Instrument | RS-5913 | |
| Micro Knife 4.75" 1.7 x 27 mm blade | Roboz Surgical Instrument | RS-6272 | |
| Moria Mini Perforated Spoon | Fine Science Tools | 10370-19 | |
| mouse monoclonal antibody to neuronal class III β-tubulin (TUBB3) | BioLegend | 801202 | 1:500, TUJ1 |
| Nikon Perfect Focus Eclipse Ti live cell fluorescence microscope and Elements software | Nikon | – | Differential interference contrast images and immunocytochemistry images of the cell cultures were captured with these equipments |
| Nitric Acid 90%, Fuming (Certified ACS) | Fisher Scientific | A202-212 | For rinsing coverslips |
| Olympus 1.7ml Microtubes, Clear | Genesee Scientific | 22-281 | These are the tubes that we described "1.7 mL microcentrifuge tubes" in the context. |
| Papain Dissociation System | Worthington Biochemical Corp | LK003150 | Papain solution and alubumin-ovomucoid inhibitor solution are prepared from this kit. |
| Penicillin-streptomycin (10,000 U/ml) | Thermo Fisher Scientific | 15140-122 | |
| Phosphate buffered saline (PBS) | Thermo Fisher Scientific | 10010-023 | |
| Poly D-lysin (PDL) | MilliporeSigma | A-003-E | |
| rabbit monoclonal antibody to Islet1 | Abcam | ab109517 | 1:200 |
| SMZ18 and SMZ1500 zoom stereomicroscopes with DS-Ri1 camera | Nikon | – | Dissection was performed and images of dissected embryos and tissues are captured under these fluorescence microscopes. |
| Sylgard 170 Black Silicone Encapsulant – A+B 0.9 Kg kit | Dow Corning | 1696157 | We make dissection dishes using this kit. |
| TC treated Dishes, 100 x 20 mm | Genesee Scientific | 25-202 | We make dissection dishes using this dish. |
| Thum Dressing Forceps 4.5" Serrated 2.2 mm Tip Width | Roboz Surgical Instrument | RS-8100 | |
| Transducer for LOGOQ e VET | GE Healthcare | L8-18i-RS | For ultrasound on female mice |
| Veterinary ultrasound machine | GE Healthcare | LOGOQ e VET | For ultrasound on female mice |
| Zeiss LSM 700 series laser scanning confocal microscope and Zen Software | Carl Zeiss | – | Confocal image of the embryo was captured with these equipments |
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