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Biology

CD4 + T-linfócitos Captura Usando um chip descartável microfluídicos para HIV

doi: 10.3791/315 Published: October 1, 2007

Summary

Protocol

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  1. Prepare canais microfluídicos com anti-CD4 + biotinlayted superfície.
  2. Injectar 10 ml de amostra de sangue de 5 ml / min no canal microfluídicos com bomba de seringa.
  3. Lavar o sangue de 20 ml / min para 20 min com PBS.
  4. Injectar a solução diluída de lise FACS (diluído a 10% da solução estoque com PBS) em 5ul/min por 15 min.
  5. Incubar por 15 min.
  6. Injetar CD3 + solução de anticorpos primários (diluído a 10% da solução estoque com PBS) a 5 ml / min para 20 min.
  7. Incubar por 2 horas.
  8. Lave canal com PBS, 5 ml / min para 15 min.
  9. Injectar 20% AF488 CD4 + de anticorpos, solução estoque 0,2% DAPI, e 10% CD3 + anticorpo secundário a 5 ml / min para 20 min, ao mesmo tempo.
  10. Incubar por 20 min.
  11. Lave o canal com PBS, 5 ml / min para 15 min.
  12. Contagem de células ao microscópio fluorescente:
    1. White (óptica) - Preto e branco: célula e poeira
    2. UV (DAPI) - Blue: única célula
    3. Azul (CD4 +) - Verde: linfócitos T e monócitos
    4. Verde (CD3 +) - Vermelho: linfócitos T só

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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-linfócitos Captura Usando um chip descartável microfluídicos para 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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