CD4 + T-לימפוציטים לכידת באמצעות שבב Microfluidic פנויה ל-HIV
Protocol
הכן ערוץ microfluidic עם משטח + אנטי CD4 biotinlayted. הזרק 10 מ"ל של דם מדגם ב 5 מ"ל / דקה לתוך התעלה microfluidic עם משאבת מזרק. לשטוף את הדם 20 מ"ל / דקה במשך 20 דקות עם PBS. הזרק פתרון FACS …
Materials
| Material Name | Tipo | 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. |
Citar este artigo
Moon, S. J., Lin, R., Demirci, U. CD4+ T-Lymphocyte Capture Using a Disposable Microfluidic Chip for HIV. J. Vis. Exp. (8), e315, doi:10.3791/315 (2007).