Observation Holm Funktion og Holm-Immune Cell interaktioner i Levende bugspytkirtel væv skiver
Summary
Denne undersøgelse præsenterer anvendelsen af levende bugspytkirtelvæv skiver til studiet af holm fysiologi og holm-immune celle interaktioner.
Abstract
Levende bugspytkirtelvæv skiver giver mulighed for undersøgelse af holm fysiologi og funktion i forbindelse med en intakt holm mikromiljø. Skiver er fremstillet af levende menneske og mus bugspytkirtelvæv indlejret i agarose og skæres ved hjælp af en vibratom. Denne metode gør det muligt for vævet at opretholde levedygtighed og funktion ud over at bevare underliggende patologier som type 1 (T1D) og type 2 diabetes (T2D). Skivemetoden muliggør nye retninger i studiet af bugspytkirtlen gennem vedligeholdelse af de komplekse strukturer og forskellige intercellulære interaktioner, der omfatter det endokrine og eksokrine væv i bugspytkirtlen. Denne protokol viser, hvordan man udfører farvning og time-lapse mikroskopi af levende endogene immunceller i bugspytkirtlen skiver sammen med vurderinger af holmen fysiologi. Endvidere kan denne tilgang raffineres til at skelne immuncellepopulationer, der er specifikke for holmcelleantigener ved hjælp af store histokompatibilitetskomplekser-multimer reagenser.
Introduction
Inddragelse af bugspytkirtlen er patognomonisk til sygdomme som pancreatitis, T1D, og T2D1,2,3. Undersøgelsen af funktion i isolerede holme indebærer normalt fjernelse af holmene fra deres omgivende miljø4. Den levende bugspytkirtelvæv skive metode blev udviklet for at give mulighed for undersøgelse af bugspytkirtelvæv samtidig opretholde intakt holm mikromiljø og undgå brugen af stressende holm isolationsprocedurer5,6,7. Bugspytkirtelvæv skiver fra human donorvæv er blevet anvendt med succes til at studere T1D og har påvist processer af beta celle tab og dysfunktion ud over immuncelle infiltration8,9,10,11,12,13. Den levende bugspytkirtelvæv skive metode kan anvendes til både mus og human bugspytkirtelvæv5,6,8. Humane bugspytkirtelvæv skiver fra organdonor væv opnås gennem et samarbejde med netværket for bugspytkirtel organdonorer med diabetes (nPOD). Museskiver kan genereres fra en række forskellige musestammer.
Denne protokol vil fokusere på ikke-fede diabetisk-rekombination aktiverende gen-1-null (NOD. Rag1-/-) og T-celle receptor transgen (AI4) (NOD. Rag1-/-. AI4 α/β) musestammer. NIKKE. Rag1-/- mus er ude af stand til at udvikle T- og B-celler på grund af en forstyrrelse i det rekombineringsaktiverende gen 1 (Rag1)14. NIKKE. Rag1-/-. AI4 α/β mus bruges som model for accelereret type 1-diabetes, fordi de producerer en enkelt T-celleklon, der er rettet mod en epitop af insulin, hvilket resulterer i konsekvent infiltration af holme og hurtig sygdomsudvikling15. Protokollen featured her beskriver procedurer for funktionelle og immunologiske undersøgelser ved hjælp af levende menneske og mus bugspytkirtel skiver gennem anvendelse af konfokal mikroskopi tilgange. De teknikker, der er beskrevet heri omfatter levedygtighed vurderinger, holm identifikation og placering, cytosolic Ca2 + optagelser, samt farvning og identifikation af immuncellepopulationer.
Protocol
Representative Results
Discussion
Formålet med denne protokol er at forklare dannelsen af bugspytkirtel skiver og de procedurer, der er nødvendige for at anvende skiver i funktionelle og immunologiske undersøgelser. Der er mange fordele ved at bruge levende bugspytkirtel skiver. Der er dog flere kritiske trin, der er afgørende for, at vævet forbliver levedygtigt og nyttigt under de beskrevne eksperimentprotokoller. Det er bydende nødvendigt at arbejde hurtigt. Afstanden mellem injektion af bugspytkirtlen og generering af skiverne på vibratomen ska…
Declarações
The authors have nothing to disclose.
Acknowledgements
Dette arbejde er finansieret af NIH tilskud R01 DK123292, T32 DK108736, UC4 DK104194, UG3 DK122638, og P01 AI042288. Denne forskning blev udført med støtte fra netværket for bugspytkirtel organdonorer med diabetes (nPOD; RRID:SCR_014641), et samarbejdsprojekt af type 1 diabetes sponsoreret af JDRF (nPOD: 5-SRA-2018-557-Q-R) og Leona M. & Harry B. Helmsley Charitable Trust (Grant #2018PG-T1D053). Det indhold og de synspunkter, der udtrykkes, er forfatternes ansvar og afspejler ikke nødvendigvis nPOD’s officielle opfattelse. Organ Procurement Organizations (OPO) partnering med nPOD at give forskningsressourcer er opført på http://www.jdrfnpod.org/for-partners/npod-partners/. Tak til Dr. Kevin Otto, University of Florida, for at give den vibratom, der anvendes til at generere mus skiver.
Materials
| #3 Style Scalpel Handle | Fisherbrand | 12-000-163 | |
| 1 M HEPES | Fisher Scientific | BP299-100 | HEPES Buffer, 1M Solution |
| 10 cm Untreated Culture Dish | Corning | 430591 | |
| 10 mL Luer-Lok Syringe | BD | 301029 | BD Syringe with Luer-Lok Tips |
| 27 G Needle | BD | BD 305109 | BD General Use and PrecisionGlide Hypodermic Needles |
| 35 mm coverglass-bottom Petri dish | Ibidi | 81156 | µ-Dish 35 mm, high |
| 50 mL syringe | BD | 309653 | |
| 8-well chambered coverglass | Ibidi | 80826 | µ-Slide 8 Well |
| APC anti-mouse CD8a antibody | Biolegend | 100712 | |
| BSA | Fisher Scientific | 199898 | |
| Calcium chloride | Sigma | C5670 | CaCl2 |
| Calcium chloride dihydrate | Sigma | C7902 | CaCl2 (dihydrate) |
| Compact Digital Rocker | Thermo Fisher Scientific | 88880020 | |
| Confocal laser-scanning microscope | Leica | SP8 | Pinhole = 1.5-2 airy units; acquired with 10x/0.40 numerical aperture HC PL APO CS2 dry and 20x/0.75 numerical aperture HC PL APO CS2 dry objectives at 512 × 512 pixel resolution |
| D-(+)-Glucose | Sigma | G7021 | C6H12O6 |
| ddiH2O | |||
| Dithizone | Sigma-Aldrich | D5130-10G | |
| DMSO | Invitrogen | D12345 | Dimethyl sulfoxide |
| Ethanol | Decon Laboratories | 2805 | |
| Falcon 35 mm tissue culture dish | Corning | 353001 | Falcon Easy-Grip Tissue Culture Dishes |
| FBS | Gibco | 10082147 | |
| Feather No. 10 Surgical Blade | Electron Microscopy Sciences | 7204410 | |
| fluo-4-AM | Invitrogen | F14201 | cell-permeable Ca2+ indicator |
| Gel Control Super Glue | Loctite | 45198 | |
| Graefe Forceps | Fine Science Tools | 11049-10 | |
| Hardened Fine Scissors | Fine Science Tools | 14090-09 | |
| HBSS | Gibco | 14025092 | Hanks Balanced Salt Solution |
| HEPES | Sigma | H4034 | C8H18N2O4S |
| Ice bucket | Fisherbrand | 03-395-150 | |
| Isoflurane | Patterson Veterinary | NDC 14043-704-05 | |
| Johns Hopkins Bulldog Clamp | Roboz Surgical Store | RS-7440 | Straight; 500-900 Grams Pressure; 1.5" Length |
| Kimwipes | Kimberly-Clark Professional | 34705 | Kimtech Science™ Kimwipes™ Delicate Task Wipers, 2-Ply |
| LIVE/DEAD Viability/Cytotoxicity Kit | Invitrogen | L3224 | This kit contains the calcein-AM live cell dye. |
| Low glucose DMEM | Corning | 10-014-CV | |
| Magnesium chloride hexahydrate | Sigma | M9272 | MgCl2 (hexahydrate) |
| Magnesium sulfate heptahydrate | Sigma | M2773 | MgSO4 (heptahydrate) |
| Magnetic Heated Platform | Warner Instruments | PM-1 | Platform for imaging chamber for dynamic stimulation recordings |
| Microwave | GE | JES1460DSWW | |
| Nalgene Syringe Filter | Thermo Fisher Scientific | 726-2520 | |
| No.4 Paintbrush | Michaels | 10269140 | |
| Open Diamond Bath Imaging Chamber | Warner Instruments | RC-26 | Imaging chamber for dynamic stimulation recordings |
| Oregon Green 488 BAPTA-1-AM | Invitrogen | O6807 | cell-permeable Ca2+ indicator |
| Overnight imaging chamber | Okolab | H201-LG | |
| PBS | Thermo Fisher Scientific | 20012050 | To make agarose for slice generation |
| PE-labeled insulin tetramer | Emory Tetramer Research Core | sequence YAIENYLEL | |
| Penicillin Streptomycin | Gibco | 15140122 | |
| Potassium chloride | Sigma | P5405 | KCl |
| Potassium phosphate monobasic | Sigma | P5655 | KH2PO4 |
| Razor Blades | Electron Microscopy Sciences | 71998 | For Vibratome; Double Edge Stainless Steel, uncoated |
| RPMI 1640 | Gibco | 11875093 | |
| SeaPlaque low melting-point agarose | Lonza | 50101 | To make agarose for slice generation |
| Slice anchor | Warner Instruments | 64-1421 | |
| Slice anchor (dynamic imaging) | Warner Instruments | 640253 | Slice anchor for dynamic imaging chamber |
| Sodium bicarbonate | Sigma | S5761 | NaHCO3 |
| Sodium chloride | Sigma | S5886 | NaCl |
| Sodium phosphate monohydrate | Sigma | S9638 | NaH2PO4 (monohydrate) |
| Soybean Trypsin Inhibitor | Sigma | T6522-1G | Trypsin inhibitor from Glycine max (soybean) |
| Stage Adapter | Warner Instruments | SA-20MW-AL | To fit imaging chamber for dynamic stimulation recordings on the microscope stage |
| Stage-top incubator | Okolab | H201 | |
| Stereoscope | Leica | IC90 E MSV266 | |
| SYTOX Blue Dead Cell Stain | Invitrogen | S34857 | blue-fluorescent nucleic acid stain |
| Transfer Pipet | Falcon | 357575 | Falcon™ Plastic Disposable Transfer Pipets |
| Valve Control System | Warner Instruments | VCS-8 | System for dynamic stimulation recordings |
| Vibratome VT1000 S | Leica | VT1000 S | |
| Water bath | Fisher Scientific | FSGPD02 | Fisherbrand Isotemp General Purpose Deluxe Water Bath GPD 02 |
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