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Biology

CD4 + T-linfociti Capture utilizzando un chip monouso Microfluidic per l'HIV

doi: 10.3791/315 Published: October 1, 2007

Summary

Protocol

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  1. Preparare canale microfluidica con anti-CD4 + biotinlayted superficie.
  2. Iniettare 10 ml di sangue campione a 5 ml / min nel canale di microfluidica con pompa a siringa.
  3. Lavare il sangue a 20 ml / min per 20 min con PBS.
  4. Iniettare una soluzione diluita di FACS lisante (diluito al 10% dalla soluzione madre con PBS) a 5ul/min per 15 minuti.
  5. Incubare per 15 min.
  6. Iniettare primario CD3 + soluzione di anticorpi (diluita al 10% dalla soluzione madre con PBS) a 5 ml / min per 20 min.
  7. Incubare per 2 ore.
  8. Scovare canale con PBS a 5 ml / min per 15 min.
  9. Iniettare 20% AF488 CD4 + anticorpi, 0,2% soluzione stock DAPI, e il 10% CD3 + anticorpi secondari a 5 ml / min per 20 minuti, allo stesso tempo.
  10. Incubare per 20 min.
  11. Lavare il canale con PBS a 5 ml / min per 15 min.
  12. Contare le cellule al microscopio a fluorescenza:
    1. Bianco (ottica) - Bianco e nero: cellulare e polvere
    2. UV (DAPI) - Blu: cell solo
    3. Blu (CD4 +) - Verde: T-linfociti e monociti
    4. Verde (CD3 +) - Rosso: T-linfociti solo

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Materials

Name Type Company Catalog Number Comments
PDMS microchannel Fabricated by SU-8 mold
Blood Donated blood from normal human subject. Mass General Hospital, 10 X high density WBC or Brigham and Women’s Hospital.
PBS Buffer Invitrogen 14190 1x Dulbecco’s Phosphated Buffered Saline.
FACS lysing solution BD Biosciences 349202 Diluted to 10% from stock solution with PBS.
Anti-CD4+ biotinlayted Ab Beckman Coulter Inc. IM0704 Surface prepared with Anti-CD4+ biotinlayted Ab, diluted to 10% from stock solution with PBS/BSA after mixing with 0.2mg stock and 1ml deionized water.
goat anti-hCD3delta pAB Santa Cruz Biotechnology, Inc. sc-1128 Diluted to 10% from stock solution with PBS
rabbit anti-goat IgG PE Santa Cruz Biotechnology, Inc. sc-3755 Secondary Ab fro anti-CD3 Ab, diluted to 10% from stock solution with PBS
anti-human CD4+ BD Biosciences 557695 Alexa Fluor 488 labeled, diluted to 20% from stock solution with PBS.
DAPI Invitrogen D1306 4’,6-dianidino-2-phenylindole, dihydrochloride solution, diluted to 0.2% from stock solution with PBS.

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CD4 + T-linfociti Capture utilizzando un chip monouso Microfluidic per l'HIV
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Cite this Article

Moon, S. J., Lin, R., Demirci, U. CD4+ T-Lymphocyte Capture Using a Disposable Microfluidic Chip for HIV. J. Vis. Exp. (8), e315, doi:10.3791/315 (2007).More

Moon, S. J., Lin, R., Demirci, U. CD4+ T-Lymphocyte Capture Using a Disposable Microfluidic Chip for HIV. J. Vis. Exp. (8), e315, doi:10.3791/315 (2007).

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