CD4 + T-linfociti Capture utilizzando un chip monouso Microfluidic per l'HIV

Protocol

Preparare canale microfluidica con anti-CD4 + biotinlayted superficie. Iniettare 10 ml di sangue campione a 5 ml / min nel canale di microfluidica con pompa a siringa. Lavare il sangue a 20 ml / min per 20 min con PBS. Iniettare una soluzione diluita di FACS lisante (diluito al 10% dalla soluzione madre con PBS) a 5ul/min per 15 minuti. Incubare per 15 min. Iniettare primario CD3 + soluzione di anticorpi (diluita al 10% dalla soluzione madre con PBS) a 5 ml / min p…

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.