CD4 + T淋巴细胞艾滋病毒的捕获使用一次性微流体芯片

Summary

Protocol

1. 准备与抗CD4 ⁺ biotinlayted表面微流体通道。
2. 10毫升的样品血液微流体通道注射泵，注射5毫升/分钟。
3. 在20毫升/分钟为20分钟，用PBS冲洗出来的血。
4. 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％）。
5. 孵育15分钟。
6. 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％） 。
7. 孵育2小时。
8. 在5毫升/分，持续15分钟，用PBS冲洗通道。
9. 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。
10. 孵育20分钟。
11. 在5毫升/分，持续15分钟，用PBS冲洗通道。
12. 荧光显微镜下细胞计数：
    1. 白色（光） - 黑色和白色：细胞和灰尘
    2. 仅紫外线（DAPI） - 蓝色：细胞
    3. 蓝（CD4 ^+） -绿色：T -淋巴细胞和单核细胞
    4. 仅绿色（CD3 ^+） -红：T淋巴细胞

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.