Protocol
- 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
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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. |