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[Bio101] Subcutaneous Injection of Tumor Cells
[Bio101] 肿瘤细胞的皮下注射   

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Abstract

Growth of cells in the subcutaneous space of immunocompromised mice is a common method for assaying tumorigenic potential in vivo. This technique is also used to assess the effects of therapeutic interventions on cancer cell lines.

Materials and Reagents

  1. Tumor cells
  2. CB17 scid/scid mice
  3. Trypsin (Life Technologies, InvitrogenTM, catalog number: 25300-054 )
  4. DMEM (Life Technologies, InvitrogenTM, catalog number: 11965-092 )
  5. Fetal bovine serum (FBS) (Life Technologies, InvitrogenTM, catalog number: 16000-044 )
  6. Phosphate buffered saline (PBS)
  7. Trypan blue (Life Technologies, InvitrogenTM, catalog number: 15250-061 )
  8. Isoflurane (usually purchased through animal facility at institution)
  9. Matrigel (BD Biosciences, catalog number: 356234 )
  10. DMEM cultural medium (see Recipes)

Equipment

  1. Centrifuges
  2. Insulin syringe
  3. Hemocytometer
  4. Cell culture hood
  5. Incubator
  6. Microscope
  7. Small gauge
  8. Eppendorf tube

Procedure

  1. Remove growth medium from cells and wash with 5 ml of PBS.
  2. Aspirate PBS, add 2 ml of trypsin and incubate for 5 min, or until cells have detached, at 37 °C.
  3. Quench trypsin by adding at least 3 volumes of 10% FBS containing medium.
  4. Pellet cells by centrifugation for 5 min at 1,100 rpm and 37 °C.
  5. Aspirate medium, wash cells with 10 ml sterile PBS, mix well with pipette and save 50 μl aliquot of cells for counting.
  6. Pellet cells by centrifugation for 5 min at 1,100 rpm and 37 °C.
  7. While cells are spinning, add 50 μl of trypan blue to saved aliquot, mix well and count cells using a hemocytometer.
    Note: Dark blue cells are dead and should not be counted.
  8. Aspirate PBS, resuspend cells in fresh PBS to a concentration 1 x 106 cells/100 μl and transfer cells to a sterile Eppendorf tube.
    Note: The cell number required depends upon the aggressiveness of the tumor cells and can vary by an order of magnitude.
  9. (Optional) Add an equal volume of thawed Matrigel to cells and mix carefully with pipette. Important, Matrigel should be thawed on ice because it will solidify at room temperature.
    Note: Matrigel provides a favorable environment for less aggressive cells to grow and is often used.
  10. Slowly pull up 100 μl of cells alone or 200 μl of cell/matrigel mixture using an insulin syringe.
    Note: Cells can be damaged by the small gauge of the insulin needle; however, insulin syringes provide a more accurate volume measurement. If significant death is observed, 1 ml syringes with 22 gauge needles can be substituted. Place cell containing syringes on ice to prevent matrigel from polymerizing.
  11. Inject 1 x 106 cells into the flanks of immune deficient mice, preferably CB17 scid/scid. To do this, pinch the skin of the mouse between your index finger and thumb and pull the skin away from the body of the mouse.
  12. Inject slowly and evenly into the pouch created by your fingers, creating a single bubble of cells beneath the skin and avoiding too much spread of the cells.
  13. Anesthetizing the mice using isoflurane makes the injection process significantly less stressful for the both the mice and the researcher.

Recipes

  1. DMEM cultural medium
    Supplemented with 10% FBS

简介

免疫受损小鼠皮下空间中细胞的生长是测定体内致瘤潜力的常用方法。 这种技术也用于评估治疗干预对癌细胞系的影响。

材料和试剂

  1. 肿瘤细胞
  2. CB17 scid/scid小鼠
  3. 胰蛋白酶(Life Technologies,Invitrogen TM,目录号:25300-054)
  4. DMEM(Life Technologies,Invitrogen TM,目录号:11965-092)
  5. 胎牛血清(FBS)(Life Technologies,Invitrogen TM ,目录号:16000-044)
  6. 磷酸盐缓冲盐水(PBS)
  7. 台盼蓝(Life Technologies,Invitrogen TM ,目录号:15250-061)
  8. 异氟烷(通常通过动物设施在机构购买)
  9. Matrigel(BD Biosciences,目录号:356234)
  10. DMEM文化媒介(见配方)

设备

  1. 离心机
  2. 胰岛素注射器
  3. 血细胞计数器
  4. 细胞培养罩
  5. 孵化器
  6. 显微镜
  7. 小规格
  8. Eppendorf管

程序

  1. 从细胞中取出生长培养基,用5ml PBS洗涤
  2. 吸出PBS,加入2ml胰蛋白酶并孵育5分钟,或直到细胞在37℃分离。
  3. 通过加入至少3体积的含10%FBS的培养基淬灭胰蛋白酶
  4. 通过在1,100rpm和37℃下离心5分钟来沉淀细胞
  5. 吸出培养基,用10ml无菌PBS洗涤细胞,用移液管充分混合,保存50μl等分的细胞用于计数。
  6. 通过在1,100rpm和37℃下离心5分钟来沉淀细胞
  7. 当细胞旋转时,加入50μl台盼蓝保存的等分试样,混合好,使用血细胞计数器计数细胞。
    注意:深蓝色的细胞已死亡,不应计算在内。
  8. 吸出PBS,将细胞在新鲜PBS中重悬至浓度为1×10 6个细胞/100μl,并将细胞转移至无菌的Eppendorf管中。
    注意:所需的细胞数量取决于肿瘤细胞的侵袭性,并且可能有一个数量级的变化。
  9. (可选)向细胞中加入等体积的解冻的Matrigel,并用移液管小心混匀。重要的是,Matrigel应该在冰上解冻,因为它会在室温下固化。
    注意:Matrigel为较不侵略的细胞生长提供了有利的环境,并且经常被使用。
  10. 使用胰岛素注射器缓慢地提起100微升的细胞或200微升的细胞/matrigel混合物 注意:细胞可能被胰岛素针的小规格损坏;然而,胰岛素注射器提供更准确的体积测量。如果观察到显着死亡,可以用22号针头替换1ml注射器。将含有注射器的细胞置于冰上以防止基质胶聚合。
  11. 将1×10 6个细胞注入免疫缺陷小鼠的侧腹,优选CB17 scid/scid。要做到这一点,捏在鼠标的食指和拇指之间的皮肤,拉动皮肤远离鼠标的身体。
  12. 缓慢均匀地注入由您的手指创建的袋,创造一个单一的泡沫细胞在皮肤下,并避免太多的细胞传播。
  13. 使用异氟烷麻醉小鼠使得注射过程对于老鼠和研究者来说显着减轻压力。

食谱

  1. DMEM培养基
    补充10%FBS
  • English
  • 中文翻译
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Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.
引用:Reuter, J. (2011). Subcutaneous Injection of Tumor Cells. Bio-protocol Bio101: e166. DOI: 10.21769/BioProtoc.166;
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emilia
What can happen if you inject the tumor cells in complete medium? Will this affect the experiment?
Thank you!
7/10/2014 11:36:48 PM Reply
Jason Reuter

You should be consistent between, but I expect the experiment will work. It should also be acknowledged that tumor growth may depend on serum and medium.

7/10/2014 11:49:13 PM


emilia

In which way may the tumor growth depend on the serum? Will not it be drain out if it is in small volum(100ul)? Thx

7/10/2014 11:59:23 PM


Jason Reuter

The fluid will be absorbed. However, it may support the cells initially. My only point is that you haven't strictly tested the cells' ability to grow tumors independent of additional supplements.

7/11/2014 12:28:01 AM


emilia

ok. Thank you very much for your advice and quick reply.

7/11/2014 12:35:30 AM