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用Optiprep分离人和其它灵长类血液多叶核白细胞

阅读数:429次  更新日期:2021-05-24   发布日期:2021-05-24

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:

  1. 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。

  1. 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。

  1. 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

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:

 

名称

规格

品牌

货号

Optiprep分离液

250ml

Axis-Shield/Alere Technologies AS

1114542

红细胞裂解液

100ml

天津灏洋/TBD

NH4CL2009

聚蔗糖400Ficoll 400

100g

BioFroxx

1345GR100

HEPES, free acid

100g

碧云天

ST092-100g

EDTA (≥99%, BioPremium)

250g

碧云天

ST1303-250g

 参考资料:

  1. Isolation of human polymorphonuclear leukocytes (granulocytes) from aleukocyte rich plasma in a discontinuous iodixanol gradient (Axis-Shield/Alere Technologies AS厂家的OptiPrep Application Sheet C12)
  2. Mononuclear cells, monocytes and polymorphonuclear leukocytes(Axis-Shield/Alere Technologies AS厂家的OptiPrep Reference List RC01)
  3. Yuan, Z-N, Tolo, K., Schenck, K. and Helgeland, K. (1999) Increased levels of soluble Fc receptor III in gingival fluid periodontal lesions Oral Microbiol. Immunol., 14, 172-175
  4. Hudgens, J., Langkamp-Henken, B., Stechmiller, J.K., Herrlinger-Garica, K.A. and Nieves, C. (2005) Immune function is impaired with a mini nutritional assessment score indicative of malnutrition in nursing home elders with pressure ulcers J. Parenter. Enteral Nutr. 28, 416-422
  5. Chiu, H-C., Liang, J-S., Wang, J-S. and Lu, J-F. (2006) Mutational analyses of Taiwanese kindred with X-linked adrenaleukodystrophy Pediatr. Neurol., 35, 250-256
  6. Channon, J.Y., Seguin, R.M. and Kasper, L. (2000) Differential infectivity and division of Toxoplasma gondii in human peripheral blood leukocytes Infect. Immun., 68, 4822-4826
  7. Channon, J.Y., Miselis, K.A:, Minns, L.A, Dutta, C. and Kasper, L.H. (2002) Toxoplasma gondii induces granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor secretion by human fibroblasts: implications for neutrophil apoptosis Infect. Immun., 70, 6048-6057
  8. Shen, L., Fahey, J.V., Hussey, S.B., Asin, S.N., Wira, C.R. and Fanger, M.W. (2004) Synergy between IL-8 and GMCSF in reproductive tract epithelial cell secretions promotes enhanced neutrophil chemotaxis Cell. Immunol., 230, 23-
  9. Shen, L., Smith, J.M., Shen, Z., Hussey, S.B., Wira, C.R. and Fanger, M.W. (2006) Differential regulation of neutrophil chemotaxis to IL-8 and fMLP by GM-CSF: lack of direct effect of oestradiol Immunology, 117, 205-212
  10. Smith, J.M., Wira, C.R., Fanger, M.W. and Shen, L. (2006) Human fallopian tube neutrophils – a distinct phenotype from blood neutrophils Am. J. Reprod. Immunol., 56, 218-229
  11. Shen, L., Smith, J.M., Shen, Z., Eriksson, M., Sentman, C. and Wira, C.R. (2007) Inhibition of human neutrophil degranulation by transforming growth factor-1 Clin. Exp. Immunol., 149, 155-161
  12. Smith, J.M., Shen, Z., Wira, C.R., Fanger, M.W. and Shen, L. (2007) Effects of menstrual cycle status and gender on human neutrophil phenotype Am. J. Reprod. Immunol., 58, 111-119
  13. Pioli, P.A., Jensen, A.L., Weaver, L.K., Amiel, E., Shen, Z., Shen, L., Wira, C.R. and Guyre, P.M. (2007) Estradiol attenuates lipopolysaccharide-induced CXC ligand 8 production by human peripheral blood monocytes J. Immunol., 179, 6284-6290
  14. Lau, M., Vayntrub, T., Grumet, F.C., Lowsky, R., Strober, S., Hoppe, R., Larson, M., Holm, B., Reitz, B. and Borie, D (2004) Short tandem repeat analysis to monitor chimerism in Macaca Fasicularis Am. J. Transplant., 4, 1543-1548