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 solution
2％台盼蓝染液
(3) Hank’s balanced salt solution (HBSS) or RPMI-1640 medium
Hank’s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 Hank’s
balanced salt solution (HBSS)
加入等体积HBSS或PBS等倍稀释血液。

(3) Pipette 2ml 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 layers are found from top to bottom:
室温2000r／min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1~2×106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，5~10min后取样作湿片高倍镜检。

活细胞不着色，死细胞染成蓝色。

Over 95% of mononuclear cells obtained are viable.
计数200个细胞，计算活细胞百分率，一般活性应在95％以上。