Summary
Protocol
- 准备与抗CD4 + biotinlayted表面微流体通道。
- 10毫升的样品血液微流体通道注射泵,注射5毫升/分钟。
- 在20毫升/分钟为20分钟,用PBS冲洗出来的血。
- 15分钟,在5ul/min注入稀释的流式细胞仪,裂解液(从股票,用PBS液稀释至10%)。
- 孵育15分钟。
- 注射5毫升/分钟,20分钟主要的CD3 +抗体解决方案(从股票,用PBS液稀释至10%) 。
- 孵育2小时。
- 在5毫升/分,持续15分钟,用PBS冲洗通道。
- 20%AF488的CD4 +抗体,0.2%的DAPI原液,10%的中学CD3 +抗体注射5毫升/分在同一时间为20分钟。
- 孵育20分钟。
- 在5毫升/分,持续15分钟,用PBS冲洗通道。
- 荧光显微镜下细胞计数:
- 白色(光) - 黑色和白色:细胞和灰尘
- 仅紫外线(DAPI) - 蓝色:细胞
- 蓝(CD4 +) -绿色:T -淋巴细胞和单核细胞
- 仅绿色(CD3 +) -红:T淋巴细胞
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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. |