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Biology

CD4 + HIV에 대한 일회용 Microfluidic 칩을 사용하여 T - 림프구 캡처

doi: 10.3791/315 Published: October 1, 2007

Summary

Protocol

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  1. 안티 CD4 + biotinlayted 표면 microfluidic 채널을 준비합니다.
  2. 주사기 펌프와 microfluidic 채널에 5 ML / 분 샘플 혈액의 10 ML을 주사.
  3. PBS로 20 분 20 ML / 분 혈액을 밖으로 플러시.
  4. 15 분 5ul/min에 희석 외과 lysing 솔루션 (PBS로 재고 솔루션에서 10 % 희석) 주사.
  5. 15 분 알을 품다.
  6. 5 ML / 20 분 분 주요 인 경우에는 3 번 CD + 항체 솔루션 (PBS로 재고 솔루션에서 10 % 희석) 주사.
  7. 2 시간 동안 부화.
  8. 5 ML / 15 분 분 PBS와 채널을 플러시.
  9. 동시에 20 분 5 ML / 분 20 % AF488 CD4 + 항체, 0.2 % DAPI 주식 솔루션,와 10 %의 보조 인 경우에는 3 번 CD + 항체를 주사.
  10. 20 분 품어.
  11. 5 ML / 15 분 분 PBS와 채널을 플러시.
  12. 형광 현미경으로 세포를 개수 :
    1. 화이트 (광학) - 흑백 : 셀 및 먼지
    2. UV (DAPI) - 블루 : 세포에만
    3. 블루 (CD4 +) - 초록색 : T - 림프구와 단핵구
    4. 그린 (인 경우에는 3 번 CD +) - 빨간색 : T - 림프구 전용

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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 + HIV에 대한 일회용 Microfluidic 칩을 사용하여 T - 림프구 캡처
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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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