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

Streszczenie

Protokół

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

Materiały

Name	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.