Isolering af ekstracellulære vesikler fra Murine Bronchoalveolar Lavage væske ved hjælp af en ultrafiltrering centrifugering teknik
Summary
Her beskriver vi to ekstracellulære vesikel isolation protokoller, ultrafiltrering centrifugering og ultracentrifugering med tæthed gradient centrifugering, at isolere ekstracellulære vesikler fra murine bronchoalveolar lavage flydende prøver. De ekstracellulære vesikler afledt af murine bronchoalveolar lavage væske ved begge metoder er kvantificeret og karakteriseret.
Abstract
Ekstracellulære vesikler (EVs) er nyligt opdagede subcellulært komponenter, der spiller en vigtig rolle i mange biologiske signalering funktioner under fysiologiske og patologiske stater. Isolering af evt fortsætter med at være en stor udfordring på dette område, på grund af begrænsninger iboende til hver teknik. Den differentierede ultracentrifugering med tæthed gradient centrifugering metode er et almindeligt anvendte tilgang og anses for at være den gyldne standard procedure for EV isolation. Men denne fremgangsmåde er tidskrævende, arbejdskrævende, og generelt resulterer i lav skalerbarhed, der ikke kan være egnet til små-volumen prøver såsom bronchoalveolar lavage væske. Vi påvise, at en ultrafiltrering centrifugering isolation metode er simpel og tid – og labor-effektive endnu giver en høj inddrivelse udbytte og renhed. Vi foreslår, at denne isolation metode kunne være en alternativ tilgang, der er egnet til EV isolation, særlig for små-volumen biologiske prøver.
Introduction
Exosomes er den mindste delmængde af evt, 50-200 nm i diameter, og har flere biologiske funktioner på tværs af en bred vifte af signalering processer1,2,3,4,5. De regulerer cellulær og væv homøostase primært ved at lette intercellulær kommunikation gennem cargo molekyler såsom lipider, proteiner og nukleinsyrer6,7,8,9 . Et kritisk trin i EV forskning er isoleret proces. Differential ultracentrifugering (UC), med eller uden tæthed gradient centrifugering (DGC), betragtes som guldstandarden tilgang, men denne metode indebærer væsentlige begrænsninger, herunder ineffektive EV genfindingsprocent og lav skalerbarhed10 , 11 , 12, at begrænse sine bedste udnyttelse til større volumen prøver, såsom celle kultur supernatanten eller høj exosome produktion prøver. Fordele og ulemper ved andre metoder, såsom størrelse udelukkelse af ultrafiltrering eller kromatografi, immunoaffinity isolation af perler eller kolonner og mikrofluidik, er godt beskrevet, og moderne supplerende procedurer er blevet udviklet til overvinde og minimere tekniske begrænsninger i hver tilgang11,12,13,14,15. Andre har vist, at en ultrafiltrering centrifugering (UFC) med en nanoporous membran i filterenhed er en alternativ teknik, der giver tilsvarende renhed til en UC metode16,17,18. Denne teknik kunne betragtes som en af metoderne alternativ isolering.
Bronchoalveolar lavage væske (BALF) indeholder evt, som besidder mange biologiske funktioner i forskellige luftvejssygdomme19,20,21,22. At studere BALF-afledte evt indebærer nogle udfordringer på grund af invasiv af bronkoskopi procedure hos mennesker, såvel som et begrænset antal lavage væske opsving. I lille laboratoriedyr som mus, kun et par milliliter kan inddrives i normal lunge betingelser, endnu mindre i betændte eller fibrotisk lungerne23. Derfor kan indsamle et tilstrækkeligt antal BALF for EV isolation af en differentieret ultracentrifugering for downstream-programmer ikke være muligt. Isolere korrekte EV populationer er imidlertid en afgørende faktor for at studere EV biologiske funktioner. Den fine balance mellem effektivitet og virkning fortsat en udfordring i veletablerede EV isolation metoder.
I denne aktuelle undersøgelse viser vi, at en centrifugal ultrafiltrering tilgang, udnytter en 100 kDa molekylvægt cut-off (MWCO) nanomembrane filterenhed, er egnet til små-volumen biologiske modellen som BALF. Denne teknik er enkel, effektiv og giver høj renhed og skalerbarhed til støtte for studiet af BALF-afledte evt.
Protocol
Representative Results
Discussion
I de seneste årtier, har forskere skilles ude betydninger af elbiler i cellulære homøostase. Endnu vigtigere, spille evt store roller i mange sygdomsprocesser af modulerende nærliggende og fjern celler gennem deres bioaktive cargo molekyler1,21,22,26,27 , 28 , 29 , <sup class=…
Divulgations
The authors have nothing to disclose.
Acknowledgements
Arbejdet er støttet af NHLBI/NIH tilskud HL103868 (til PC) og HL137076 (til PC), den amerikanske hjerte sammenslutning licensbetaling (til PC) og Samuel Oschin omfattende Cancer Institute (SOCCI) Lung Cancer Research Award (til PC). Vi vil gerne give udtryk for vores store påskønnelse Smidt Heart Institute på Cedars-Sinai Medical Center, der giver os en Nanosight maskine for EV nanopartikel tracking analyse.
Materials
| Material | |||
| Amicon Ultra-15 centrifugal filters Ultracel-100K | Sigma-Millipore, St. Louis, MO | UFC910024 | |
| Dulbecco's Phosphate Buffered Saline (DPBS) | Corning Cellgro, Manassas, VA | 21-031-CV | |
| Sucrose | Sigma-Millipore, St. Louis, MO | EMD8550 | |
| HEPES | Research Products International, Prospect, IL | 75277-39-3 | |
| EDTA | Corning Cellgro, Manassas, VA | 46-034-CI | |
| Sodium Chloride | Sigma-Millipore, St. Louis, MO | S3014-1KG | |
| OptiPrep | Sigma-Millipore, St. Louis, MO | MKCD9753 | Density Gradient Medium |
| Ketamine | VetOne, Boise, ID | 13985-702-10 | |
| Xylazine | Akorn Animal Health, Lake Forest, IL | 59399-110-20 | |
| Syringe 1 mL | BD Syringe, Franklin Lakes, NJ | 309656 | |
| Angiocatheter 20G | BD Syringe, Franklin Lakes, NJ | 381703 | |
| Centrifuge tubes 15 mL | VWR, Radnor, PA | 89039-666 | |
| Centrifuge tubes 50 mL | Corning Cellgro, Manassas, VA | 430828 | |
| Bicinchonic acid (BCA) protein assay | Pierce, Thermo Fischer Scientific, Rockford, IL | 23235 | |
| Rabbit anti-mouse TSG101 Antibody | AbCam, Cambridge, MA | AB125011 | |
| Rat anti-mouse PE-CD63 Antibody | Biolegend, San Diego, CA | 143904 | |
| CD81 | |||
| CD9 | |||
| Anti-rabbit IgG, HRP-linked antibody | Cell Signaling Technology, Danvers, MA | 7074S | |
| 4x LDS | |||
| 10x Reducing agent (Bolt) | |||
| 10x Lysis buffer (Bolt) | Cell Signaling Technology, Danvers, MA | ||
| Bolt 4-12% Bis-Tris Plus acrylamide gel | Invitrogen, Thermo Fisher Scientific, Waltham, MA | NW04120 | |
| iBlot 2 Nitrocellulose mini stacks | Invitrogen, Thermo Fisher Scientific, Waltham, MA | IB23002 | |
| Chemiluminescent HRP antibody detection reagent HyGLO | Denville Scientific, Holliston, MA | E2400 | |
| Ultracentrifuge tubes 17 mL | Beckman Coulter, Pasadena, CA | 337986 | |
| Ultracentrifuge tubes 38.5 mL | Beckman Coulter, Pasadena, CA | 326823 | |
| Corning SFCA Syringe Filters 0.2 µm pore | Thermo Fisher Scientific, Waltham, MA | 09-754-13 | |
| Equipment | |||
| Centrifuge | Eppendorf, Hamburg, Germany | – | |
| Ultracentrifuge | Beckman Coulter, Pasadena, CA | – | |
| Nanosight (NS300) | Malvern, Worcestershire, UK | – | To measure particle size distribution and particle concentration |
| MACSQuant Analyzer 10 flow cytometer | Miltenyi Biotec, Bergisch Gladbach, Germany | – | |
| iBlot Transfer Apparatus | Thermo Fischer Scientific, Waltham, MA | – | |
| Bio-Rad ChemiDoc MP Imaging System | Bio-Rad, Hercules, CA | ||
| FlowJo v. 10 | Analysis software |
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