CD4+ T-Lymphocyte Capture Using a Disposable Microfluidic Chip for HIV

Biology

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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).

Abstract

Protocol

  1. Prepare microfluidic channel with anti-CD4+ biotinlayted surface.
  2. Inject 10 ml of sample blood at 5 ml/min into the microfluidic channel with syringe pump.
  3. Flush out the blood at 20 ml/min for 20 min with PBS.
  4. Inject diluted FACS lysing solution (diluted to 10% from stock solution with PBS) at 5ul/min for 15 min.
  5. Incubate for 15 min.
  6. Inject primary CD3+ antibody solution (diluted to 10% from stock solution with PBS) at 5 ml/min for 20 min.
  7. Incubate for 2 hours.
  8. Flush out channel with PBS at 5 ml/min for 15 min.
  9. Inject 20% AF488 CD4+ antibody, 0.2% DAPI stock solution, and 10% secondary CD3+ antibody at 5 ml/min for 20 min at the same time.
  10. Incubate for 20 min.
  11. Flush out the channel with PBS at 5 ml/min for 15 min.
  12. Count cells under fluorescent microscope:
    1. White (optical) - Black and white: cell and dust
    2. UV(DAPI) - Blue: cell only
    3. Blue(CD4+) - Green: T-lymphocyte and monocyte
    4. Green(CD3+) - Red: T-lymphocyte only

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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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Comments

2 Comments

  1. Could you elaborate on your staining and image capture procedures?

    Reply
    Posted by: Anonymous
    October 9, 2007 - 11:39 AM
  2. Could you elaborate on your staining and image capture procedures?

    Reply
    Posted by: Anonymous
    October 9, 2007 - 1:29 PM

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