T CD4 + des lymphocytes capturer en utilisant une puce jetable microfluidiques pour le VIH

Protocol

Préparer canal microfluidique avec des anti-CD4 + de surface biotinlayted. Injecter 10 ml de l'échantillon sanguin à 5 ml / min dans le canal microfluidique avec pompe seringue. Rincer le sang à 20 ml / min pendant 20 min avec du PBS. Injecter une solution diluée d'FACS lyse (dilué à 10% de la solution stock avec PBS) à 5ul/min pendant 15 min. Incuber pendant 15 min. Injecter CD3 + solution d'anticorps primaire (dilué à 10% de solution stock a…

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.