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Isolation of Mononuclear Cell Populations from Ovarian Carcinoma Ascites

从卵巢癌腹水中分离单核细胞群

Christina Wefers Christina Wefers
GB Ghaith Bakdash
Meritxell Moreno Martin Meritxell Moreno Martin
TB Tjitske Duiveman-de Boer
Ruurd Torensma Ruurd Torensma
LM Leon F.A.G. Massuger
IV I. Jolanda M. de Vries

发布: 2017年04月05日第7卷第7期 DOI: 10.21769/BioProtoc.2219 浏览次数: 12481

评审: Lee-Hwa TaiJalaj GuptaRalph Bottcher

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Abstract

Ovarian cancer is one of the most fatal tumors in women. Due to a lack of symptoms and adequate screening methods, patients are diagnosed at advanced stages with extensive tumor burden (Jelovac and Armstrong, 2011). Interestingly, ovarian cancer metastasis is generally found within the peritoneal cavity rather than other tissues (Lengyel, 2010; Tan et al., 2006). The reason behind this tissue tropism of the peritoneal cavity remains elusive. A prominent feature of this selectivity is ascites, the accumulation of fluid within the peritoneal cavity, containing, amongst others, immune cells, tumor cells and various soluble factors that can be involved in the progression of ovarian cancer (Kipps et al., 2013). The protocol described here is used to isolate mononuclear cells from ascites to study the functionality of the immune system within the peritoneal cavity.

Keywords: Ovarian cancer (卵巢癌) search Ascites (腹水) search Fluorescent activated cell sorting (荧光激活细胞分选) search Mononuclear cells (单核细胞) search Dendritic cells (树突状细胞) search Monocytes (单核细胞) search Myeloid-derived suppressive cells (髓源性抑制细胞) search BDCA1+CD14+ cells (BDCA1+CD14+细胞) search

Background

Gradient centrifugation using LymphoprepTM is a standard protocol to isolate peripheral blood mononuclear cells (PBMCs). We slightly adjusted the protocol, regarding the sample preparation and amount of washing steps, in order to isolate mononuclear cells from ascites.

Materials and Reagents

  1. 50 ml tubes (Greiner Bio One International, catalog number: 227261 )
  2. Cell strainer 100 μm (Corning, Falcon®, catalog number: 352360 )
  3. 5 ml pipette (VWR, catalog number: VWR612-3702 )
  4. 10 ml pipette (VWR, catalog number: VWR612-3700 )
  5. 25 ml pipette (VWR, catalog number: VWR612-3697 )
  6. 225 ml tubes (Corning, Falcon®, catalog number: 352075 )
  7. 5 ml polypropylene round-bottom tube (Corning, Falcon®, catalog number: 352063 )
  8. Pre-separation filter 30 μm (Miltenyi Biotec, catalog number: 130-041-407 )
  9. Türk’s solution (EMD Millipore, catalog number: 1092770100 )
  10. LymphoprepTM (Alere Technologies, Axis-Shield, catalog number: 1114547 )
  11. Trypan blue stain (Thermo Fisher Scientific, GibcoTM, catalog number: 15250061 )
  12. FcR blocking reagent (Miltenyi Biotec, catalog number: 120-000-442 )
  13. Antibodies
    1. Anti-CD45-V450 (BD, BD Biosciences, catalog number: 560367 )
    2. Anti-CD19-FITC (Dako Cytomation, catalog number: F0768 )
    3. Anti-CD20-FITC (BD, BD Biosciences, catalog number: 345792 )
    4. Anti-CD56-FITC (BD, BD Biosciences, catalog number: 345811 )
    5. Anti-CD1c-PE (BDCA1) (Miltenyi Biotec, catalog number: 130-090-508 )
    6. Anti-CD14-PerCP (BD, BD Biosciences, catalog number: 345786 )
    7. Anti-HLA-DR-PE-Cy7 (BD, BD Biosciences, catalog number: 335830 )
    8. Anti-CD4-APC-Cy7 (BD, BD Biosciences, catalog number: 557871 )
    9. Anti-CD3-BV510 (BD, BD Biosciences, catalog number: 563109 )
  14. Ethylenediaminetetraacetic acid (EDTA) (EMD Millipore, catalog number: 1084211000 )
  15. Bovine serum albumin (BSA) (Roche Diagnostics, catalog number: 10735108001 )
  16. Phosphate-buffered saline (PBS) (Braun Melsungen, catalog number: 362 3140 )
  17. X-VIVO 15 (Lonza, catalog number: BE02-060Q )
  18. Antibiotic/Antimycotic (AA) (Thermo Fisher Scientific, GibcoTM, catalog number: 15240062 )
  19. Human serum (HS) (Bloodbank Rivierenland)
  20. 0.5 M EDTA (see Recipes)
  21. 10% BSA (see Recipes)
  22. Diluting buffer (see Recipes)
  23. Wash buffer (see Recipes)
  24. Staining buffer (see Recipes)
  25. Medium (see Recipes)

Equipment

  1. Centrifuge (Hettich Instruments, model: Rotanta 460R )
  2. Cell sorter (BD, model: FACS Aria II)
  3. Magnetic stirrer (Heidolph instruments, model: MR Hei-Mix S )

Procedure

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文章信息

版权信息

© 2017 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Wefers, C., Bakdash, G., Moreno Martin, M., Duiveman-de Boer, T., Torensma, R., Massuger, L. F. and de Vries, I. J. M. (2017). Isolation of Mononuclear Cell Populations from Ovarian Carcinoma Ascites. Bio-protocol 7(7): e2219. DOI: 10.21769/BioProtoc.2219.

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分类

细胞生物学 > 细胞分离和培养 > 细胞分离

细胞生物学 > 基于细胞的分析方法 > 流式细胞术

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