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Biology

CD4 + T淋巴细胞艾滋病毒的捕获使用一次性微流体芯片

doi: 10.3791/315 Published: October 1, 2007

Summary

Protocol

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  1. 准备与抗CD4 + biotinlayted表面微流体通道。
  2. 10毫升的样品血液微流体通道注射泵,注射5毫升/分钟。
  3. 在20毫升/分钟为20分钟,用PBS冲洗出来的血。
  4. 15分钟,在5ul/min注入稀释的流式细胞仪,裂解液(从股票,用PBS液稀释至10%)。
  5. 孵育15分钟。
  6. 注射5毫升/分钟,20分钟主要的CD3 +抗体解决方案(从股票,用PBS液稀释至10%) 。
  7. 孵育2小时。
  8. 在5毫升/分,持续15分钟,用PBS冲洗通道。
  9. 20%AF488的CD4 +抗体,0.2%的DAPI原液,10%的中学CD3 +抗体注射5毫升/分在同一时间为20分钟。
  10. 孵育20分钟。
  11. 在5毫升/分,持续15分钟,用PBS冲洗通道。
  12. 荧光显微镜下细胞计数:
    1. 白色(光) - 黑色和白色:细胞和灰尘
    2. 仅紫外线(DAPI) - 蓝色:细胞
    3. 蓝(CD4 +) -绿色:T -淋巴细胞和单核细胞
    4. 仅绿色(CD3 +) -红:T淋巴细胞

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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淋巴细胞艾滋病毒的捕获使用一次性微流体芯片
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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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