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Biology

CD4 + T-lymfocyter ta med en engångs Mikroflödessystem Chip för HIV

doi: 10.3791/315 Published: October 1, 2007

Summary

Protocol

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  1. Förbered mikroflödessystem kanal med anti-CD4 + biotinlayted yta.
  2. Injicera 10 ml prov blod vid 5 ml / min i mikroflödessystem kanal med sprutpump.
  3. Spola ut blodet på 20 ml / min i 20 minuter med PBS.
  4. Injicera utspädd FACS Lyseringslösning (utspätt till 10% från stamlösningen med PBS) i 5ul/min i 15 min.
  5. Inkubera i 15 minuter.
  6. Injicera primära CD3 + antikropp lösning (utspädd till 10% från stamlösningen med PBS) vid 5 ml / min i 20 min.
  7. Inkubera i 2 timmar.
  8. Spola ut kanal med PBS vid 5 ml / min i 15 min.
  9. Injicera 20% AF488 CD4 + antikropp, 0,2% DAPI stamlösning, och 10% sekundär CD3 + antikroppar vid 5 ml / min i 20 minuter på samma gång.
  10. Inkubera i 20 min.
  11. Spola kanalen med PBS vid 5 ml / min i 15 min.
  12. Räkna celler i fluorescerande mikroskop:
    1. Vit (optisk) - Svart och vit: Cell-och damm
    2. UV (DAPI) - Blå: cell bara
    3. Blå (CD4 +) - Grön: T-lymfocyter och monocyter
    4. Grön (CD3 +) - Röd: T-lymfocyter bara

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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-lymfocyter ta med en engångs Mikroflödessystem Chip för 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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