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

doi:10.3791/315

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

Sang Jun Moon¹, Richard Lin², Utkan Demirci¹
¹Bio-Acoustic-MEMS Laboratory in Medicine (BAMM), HST-Center for Bioengineering, Brigham and Women's Hospital, ²Massachusetts Institute of Technology


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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:
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


			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		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.


			Cite this Article

Moon SJ, Lin R, Demirci U (2007). CD4+ T-Lymphocyte Capture Using a Disposable Microfluidic Chip for HIV. JoVE. 8. http://www.jove.com/index/details.stp?id=315, doi: 10.3791/315


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10/09/2007 11:39:42 AM

LabGirl responded with a statement of type: Neutral

Could you elaborate on your staining and image capture procedures?

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10/09/2007 1:29:29 PM

LabGirl responded with a statement of type: Question

Could you elaborate on your staining and image capture procedures?

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Keywords

Cellular Biology, Issue 8, microfluidic, blood, diagnostics, bioengineering, HIV, Translational Research

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10/01/2007

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8327

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doi: 10.3791/315

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About Jove

Journal of Visualized Experiments (JoVE) is an online research journal employing visualization to increase reproducibility and transparency in biological sciences.

ISSN 1940-087X

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