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Peer-reviewed

Determining Leukocyte Origins Using Parabiosis in the PyMT Breast Tumor Model

采用联体模型在PyMT乳腺肿瘤模型中测定白血球起源

Ruth A. Franklin Ruth A. Franklin
ML Ming O. Li

发布: 2015年08月20日第5卷第16期 DOI: 10.21769/BioProtoc.1567 浏览次数: 9948

评审: Mario CioceAnonymous reviewer(s)

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Abstract

Tumors develop in a complex microenvironment alongside numerous cell types that impact their survival. Immune cells make up a large proportion of these accessory cells and many are known to promote tumor progression. Macrophages, in particular, are associated with poor patient prognosis and are therefore potential candidates for therapeutic targeting in cancer. However, to develop successful strategies to target macrophages, it is important to clarify whether these cells are derived from blood-borne precursors or a tissue-resident population. Parabiosis, or the surgical connection of two mice resulting in a shared blood circulation, allows the distinction between these two cellular sources. Here, we describe the use of parabiosis to define cell ontogeny in a mouse model of breast cancer.

Keywords: Tumor-associated macrophages (肿瘤相关巨噬细胞) search Monocytes (单核细胞) search Breast cancer (乳腺癌) search Oncogene-driven tumor model (癌基因驱动的肿瘤模型) search

Materials and Reagents

  1. Mice: Female age- and weight-matched congenically-marked MMTV-PyMT mice [see Franklin et al. (2014) for specific strain information]
  2. Injectable anesthesia (ketamine/xylazine cocktail: 150 mg/kg/15 mg/kg)
  3. Antiseptic solution (Betadine)
  4. 70% ethanol
  5. Ophthalmic ointment (Paralube)
  6. Analgesic (Buprenorphine, 0.1 mg/kg)
  7. Non-wetting sterile water (HydroGel)
  8. Sterile saline solution (0.9% NaCl)
  9. 1x PBS
  10. 10x PBS
  11. Bovine serum albumin (Sigma-Aldrich, catalog number: A2153 )
  12. Sodium azide (Sigma-Aldrich, catalog number: S2002 )
  13. Collagenase Type III (Worthington Biochemicals, catalog number: LS004182 )
  14. Percoll (Sigma-Aldrich, catalog number: P1644 )
  15. HBSS + calcium chloride + magnesium chloride (Life Technologies, Gibco®, catalog number: 14025 )
  16. Deoxyribonuclease I from bovine pancreas (Sigma-Aldrich, catalog number: DN25 )
  17. CD45.1 APC-eFluor® 780 (eBioscience, catalog number: 47-0453-82 )
  18. CD45.2 PerCP-Cyanine5.5 (eBioscience, catalog number: 45-0454-82 )
  19. MHC Class II (I-A/I-E) FITC (eBioscience, catalog number: 11-5321-82 )
  20. CD11b Pacific Blue™ (Life Technologies, InvitrogenTM, catalog number: RM2828 )
  21. Ly-6C APC (BD Pharmingen, catalog number: 560595 )
  22. Ly-6G PE (BD Pharmingen, catalog number: 551461 )
  23. Fc block (2.4G2), (BD Pharmingen, catalog number: 553142 )
  24. RPMI 1640 (Life Technologies, Gibco®, catalog number: 11875-093 )
  25. Digestion buffer (see Recipes)
  26. 100% Percoll (see Recipes)
  27. 44% Percoll (see Recipes)
  28. 66% Percoll (see Recipes)
  29. FACS buffer (see Recipes)

Equipment

  1. Non-absorbable black nylon suture (4-0, 13mm, 3/8 circle) (Ethicon, catalog number: 1854G )
  2. Wound clip applier, wound clip remover, wound clips (Autoclip, 9 mm) (Braintree Scientific, catalog number: ACS KIT )
  3. Cotton-tipped applicators (3 inch, wood shaft) (Fisherbrand, catalog number: 23-400-105 )
  4. Sterile gauze (Fisherbrand, catalog number: 22-028-558 )
  5. Needle holder with locking mechanism (Fine Science Tools)
  6. Scissors (Fine Science Tools)
  7. Curved forceps (2 pairs) (Fine Science Tools)
  8. Razor blades
  9. Slides (Corning Incorporated, catalog number: 2948-75X25 )
  10. Heating lamp
  11. Heating pads
  12. Electrical shaver
  13. Surgical drapes/sterile pads (Fisherbrand)
  14. 6 well non-tissue culture treated plates (Falcon®)
  15. 70 micron cell strainers (Falcon®, catalog number: 352350 )
  16. 50 ml conical tubes (Falcon®)
  17. 15 ml conical tubes (Falcon®)
  18. 3 ml syringe (BD Bioscience)
  19. U-bottom 96 well plates (Falcon®)
  20. Desktop centrifuge (Sorvall)
  21. 37 °C water bath
  22. Flow cytometer (BD LSR II or similar)

Procedure

English
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文章信息

版权信息

© 2015 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Franklin, R. A. and Li, M. O. (2015). Determining Leukocyte Origins Using Parabiosis in the PyMT Breast Tumor Model. Bio-protocol 5(16): e1567. DOI: 10.21769/BioProtoc.1567.

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分类

癌症生物学 > 肿瘤免疫学 > 动物模型

免疫学 > 动物模型 > 小鼠

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