单个核细胞分离

1、Separation of Mononuclear Cells from Whole Peripheral Blood外周血单个核细胞分离Ficoll-Hypaque density gradient centrifugation聚蔗糖泛影葡胺分层液密度梯度离心法【Materials】(1) Lymphocyte Separation Medium: specific gravity 1.077±0.001g/L淋巴细胞分层液（比重为1.077±0.001g/L）(2) 2 trypan blue solution2台盼蓝染液(3) Hanks balanced salt

2、solution (HBSS) or RPMI-1640 medium Hanks液或者RPMI-1640培养基【Methods】(1) Drawing 2ml blood from main vein and mixing with 0.1 ml Heparin solution in a tube.采集静脉血2ml注入盛有0.1ml肝素的试管中，混匀。(2) Dilute the whole blood with an equal volume of Hanks balanced salt solution (HBSS)加入等体积HBSS或PBS等倍稀释血液。(3) Pipette 2ml

3、 lymphocyte separation medium into a centrifuge tube. Then, add the diluted blood sample carefully by flowing along the side of the tube on the LSM. The interface must be clear.吸取淋巴细胞分层液2ml加入离心管中，再将稀释血液小心沿管壁加至分层液上，应注意保持两者界面清晰。(4) Centrifugation at 2000rpm for 20 min at room temperature.Four distinct

4、 layers are found from top to bottom:室温2000rmin离心20min。管内可分为以下四层：(5) Harvest the band of mononuclear cells by a pipette from the tube and transfer them into a new clean tube containing 5ml HBSS.用毛细吸管轻轻吸出灰白色的单个核细胞，加入另一支已含有5mlHBSS的离心管中，混匀。(6) Centrifugation at 1500rpm for 10 min at room temperature.室温

5、下,以1500rpm离心10min，可去除混杂的血小板.(7) Discard the supernatant and repeat once.弃上清，重复洗涤一次。Resuspend the cell pellet by 1ml RPMI-1640 medium. Take count of cell numbers and adjust cell suspension to a desired concentration.用完全RPMI-1640 1ml定容，计数细胞，调整细胞悬液至所需浓度：一般每ml血可分离出12×106个单个核细胞。(8) Add a drop of 2% trypan blue solution to the tube with 0.1 ml cell suspension. Mixed and distinguish live cells (unstained) and dead cells (stained blue) after 5-10min.取0.1ml细胞悬液加1滴2%台盼蓝染液，510min后取样作湿片高倍镜检。活细胞不着