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
Materials
Material Name
Type
Company
Catalogue Number
Comment
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
Becton Dickinson
349202
Diluted to 10% from stock solution with PBS.
Anti-CD4+ biotinlayted Ab
Beckman Coulter
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
sc-1128
Diluted to 10% from stock solution with PBS
rabbit anti-goat IgG PE
Santa Cruz Biotechnology
sc-3755
Secondary Ab fro anti-CD3 Ab, diluted to 10% from stock solution with PBS
anti-human CD4+
Becton Dickinson
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.
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).