Summary
Protocol
- Prepare canal de microfluidos con anti-CD4 + biotinlayted superficie.
- Inyectar 10 ml de muestra de sangre a 5 ml / min en el canal de microfluidos con bomba de jeringa.
- Expulsar la sangre a 20 ml / min durante 20 minutos con PBS.
- Inyectar la solución diluida FACS lisis (diluido al 10% de la solución madre con PBS) a 5ul/min durante 15 minutos.
- Incubar durante 15 min.
- Inyectar primaria CD3 + solución de anticuerpos (diluido al 10% de la solución madre con PBS) a 5 ml / min durante 20 min.
- Incubar durante 2 horas.
- Enjuague con canal PBS a 5 ml / min durante 15 min.
- Inyectar 20% AF488 anticuerpos CD4 +, 0,2% de solución madre DAPI, y el 10% CD3 + secundaria de anticuerpos en 5 ml / min durante 20 minutos a la vez.
- Incubar durante 20 min.
- Limpiar los canales de PBS a 5 ml / min durante 15 min.
- Recuento de células bajo microscopio de fluorescencia:
- Blanco (óptico) - Negro y blanco: la célula y el polvo
- UV (DAPI) - Azul: células sólo
- Azul (CD4 +) - Verde: los linfocitos T y monocitos
- Verde (CD3 +) - Red: los linfocitos T sólo
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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 | BD Biosciences | 349202 | Diluted to 10% from stock solution with PBS. | |
Anti-CD4+ biotinlayted Ab | Beckman Coulter Inc. | 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, Inc. | sc-1128 | Diluted to 10% from stock solution with PBS |
rabbit anti-goat IgG PE | Santa Cruz Biotechnology, Inc. | sc-3755 | Secondary Ab fro anti-CD3 Ab, diluted to 10% from stock solution with PBS | |
anti-human CD4+ | BD Biosciences | 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. |