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

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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