Summary
Protocol
- Förbered mikroflödessystem kanal med anti-CD4 + biotinlayted yta.
- Injicera 10 ml prov blod vid 5 ml / min i mikroflödessystem kanal med sprutpump.
- Spola ut blodet på 20 ml / min i 20 minuter med PBS.
- Injicera utspädd FACS Lyseringslösning (utspätt till 10% från stamlösningen med PBS) i 5ul/min i 15 min.
- Inkubera i 15 minuter.
- Injicera primära CD3 + antikropp lösning (utspädd till 10% från stamlösningen med PBS) vid 5 ml / min i 20 min.
- Inkubera i 2 timmar.
- Spola ut kanal med PBS vid 5 ml / min i 15 min.
- Injicera 20% AF488 CD4 + antikropp, 0,2% DAPI stamlösning, och 10% sekundär CD3 + antikroppar vid 5 ml / min i 20 minuter på samma gång.
- Inkubera i 20 min.
- Spola kanalen med PBS vid 5 ml / min i 15 min.
- Räkna celler i fluorescerande mikroskop:
- Vit (optisk) - Svart och vit: Cell-och damm
- UV (DAPI) - Blå: cell bara
- Blå (CD4 +) - Grön: T-lymfocyter och monocyter
- Grön (CD3 +) - Röd: T-lymfocyter bara
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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. |