分离人血液多叶核白细胞(Polymorphonuclear Leukocytes/granulocytes,多形核细胞/多型核细胞),一般用即用型产品Polymorphprep(Axis-Shield/Alere Technologies AS,Cat No:1114683),操作简单,效果也比较满意,但对于某些人员的血液(如贫血),和血液样本从人体抽出后超过2小时时,该产品效果欠佳,这时可以考虑本方案。
本方案需要先将血液制备成富含白细胞的血浆(LRP/leukocyte-rich plasma ),再利用多叶核白细胞和单个核细胞的密度差,在离心管中依次铺上配制成的不同密度的分离液,再离心,从而在离心管中不同的界面处,分别得到多叶核白细胞和单个核细胞。该方案更为通用。具体步骤如下:
- 溶液准备:
溶液A:OptiPrep™ (60%, w/v iodixanol),使用前轻轻摇晃均匀;
溶液B:等渗盐溶液,0.85% (w/v) NaCl, 1 mM EDTA, 20 mM HEPES-NaOH, pH 7.4;
溶液C:6% (w/v) 聚蔗糖(Mr = 400-500x 10^3),在0.85% (w/v) NaCl溶液中;
溶液D:红细胞裂解液;
溶液E:1.8 (w/v) NaCl, 20 mM Hepes-NaOH, pH 7.4。
- 分离步骤
(1)向9体积新鲜的EDTA抗凝血加入1体积的溶液C;(Notes1)
(2)室温放置20-40 min,聚集的红细胞沉淀到底部,然后吸出上清,获得富白细胞血浆(leukocyte-rich plasma, LRP);
(3)配置密度分离液:1.077g/ml (5体积OptiPrep™ + 17体积溶液B)、1.090 g/ml (8体积OptiPrep™ +22体积溶液B)或1.095 g/ml (17体积OptiPrep™ + 43体积溶液B);(Notes 2)
(4)依次向离心管中加入2.5-3.0 ml的1.090 g/ml或1.095 g/ml密度分离液、相同体积(2.5-3.0 ml)的1.077 g/ml密度分离液、5 ml的LRP;(见图)
(5)水平转子,18-22℃,800×g离心25 min;
(6)此时出现两个白膜层,下层为多形核粒细胞层(PMNs),上层为单个核细胞层,用移液器小心吸取目标细胞层;(见图)
(7)使用溶液B稀释获得的PMNs悬液(1:1),250-350×g离心10min,获得PMNs;
(8)加入使用3 ml溶液D,37℃孵育7min,裂解红细胞(或者直接加入3 ml冰冷的蒸馏水,30s后加入3 ml溶液E,以裂解红细胞);
(9)250-300×g离心15 min,根据实验需要重悬细胞。
- 注意事项(Notes)
① 如果不想让细胞暴露于聚蔗糖溶液(研究表明,聚蔗糖大分子可能会吸附白细胞),可以直接在18-22℃下,200×g离心15-20min,然后取红细胞上层的血浆上清中的白细胞层,在步骤4中使用;
② 如果使用1.090 g/ml密度,少量中性粒细胞和大量嗜酸性粒细胞会通过该层沉淀;如果使用1.095 g/ml密度,几乎所有的PMNs被沉淀在该层。另外,若LRP中残余少量红细胞,使用低密度可能会污染PMNs层。
该方案已经在多篇文献中用到,如下是其中的一部分:
种属 | 密度梯度和离心参数 | 参考资料 |
人 | 碘克沙醇溶液密度 1.077+1.090g/ml,离心力 1200g离心时间 25 min | 3 |
| 碘克沙醇溶液密度 1.077+1.090g/ml,离心力 800g离心时间 30 min | 4 |
| 碘克沙醇溶液密度 1.077+1.090g/ml,离心力 600g离心时间 20 min | 5 |
| 即用型PBMC分离液+碘克沙醇溶液密度1.095g/ml,离心力400/500g离心时间 25/20min | 6-14 |
猴子 | Iodixanolsolutionsof1.077+1.095g/ml | 15 |
该方案也已用在多种其它动物血液上,如牛,豚鼠,小鼠,兔和大.等,具体文献件参考资料2.
用到的产品:
参考资料:
- Isolation of human polymorphonuclear leukocytes (granulocytes) from aleukocyte rich plasma in a discontinuous iodixanol gradient (Axis-Shield/Alere Technologies AS/Serumwerk Bernburg AG厂家的OptiPrep Application Sheet C12)
- Mononuclear cells, monocytes and polymorphonuclear leukocytes(Axis-Shield/Alere Technologies AS/Serumwerk Bernburg AG厂家的OptiPrep Reference List RC01)
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