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