搜索

Isolation of Mouse Tumor-Infiltrating Leukocytes by Percoll Gradient Centrifugation
采用Percoll密度梯度离心法分离小鼠肿瘤浸润性白细胞   

评审
匿名评审
下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

Abstract

A hallmark of cancer-associated inflammation is the infiltration of leukocytes into tumors, which is believed to be recruited by chemokines. Some infiltrating leukocytes such as macrophages often promote tumor growth by producing growth-inducing and angiogenic factors. Here, we described a method of isolating tumor-infiltrating leukocytes with Percoll density gradient, because Percoll possesses a low viscosity, a low osmolarity and no toxicity to cells. Different leukocyte populations are isolated based on their density and collected at the interface between 40% and 80% discontinuous Percoll gradient.

Materials and Reagents

  1. Mice with tumors
  2. Collagenase, type IV (Sigma-Aldrich, catalog number: C5138 )
  3. DNase, type IV (Sigma-Aldrich, catalog number: D5025 )
  4. Fungizone Antimycotic (Life Technologies, InvitrogenTM, catalog number: 15290-026 )
  5. Hank's Balanced Salt Solution (HBSS), without Calcium Chloride or Magnesium Chloride (Lonza, catalog number: 10-547F )
  6. Hyaluronidase, Type V (Sigma-Aldrich, catalog number: H6254 )
  7. Bovine Serum Albumin (BSA) (Sigma-Aldrich, catalog number: A4503 )
  8. 0.5 M EDTA (Quality Biological Inc, catalog number: qb351-027-721 )
  9. Percoll (GE Healthcare, catalog number: 17-0891-01 )
  10. Dulbecco’s Modified Eagle’s Medium High glucose with stable L-glutamine (DMEM) (Lonza, catalog number: 12-614F )
  11. Fetal Bovine Serum (FBS) (Thermo Fisher Scientific, catalog number: SH30070-03 )
  12. 10x PBS (Life Technologies, Gibco®, catalog number: 70011 )
  13. ACK lysing buffer (Lonza, catalog number: 10-548E )
  14. 10x Triple Enzyme Stock Solution (see Recipes)
  15. Wash buffer (see Recipes)
  16. 40% Percoll (see Recipes)
  17. 80% Percoll (see Recipes)

Equipment

  1. 25 ml canted Neck and Blue Plug Seal Cap, non-vented tissue culture flasks (BD Biosciences, Falcon®, catalog number: 353018 )
  2. Shaker (Lab-Line, model: 1314 )
  3. Centrifuge
  4. 100 mm Petri dish

Procedure

  1. Dissect Lewis Lung Cancer (LLC) tumors (tumor volume ~1,000 mm3/tumor, tumor weight: 0.5~0.8 g/tumor) grown on mice.
  2. Place one tumor into a 100 mm Petri dish and add 5-10 ml HBSS at room temperature (RT).
  3. Quickly mince tumors with scalpels into fragments small enough to be aspirated into a 5 ml pipette without getting stuck at RT.
  4. Transfer tumor tissue suspension into 50 ml non-vented tissue culture flasks.
  5. Rinse Petri dish with up to 40 ml HBSS and transfer the suspension into the flask. Total volume in the flask is 45 ml.
  6. Add 5 ml 10x Triple Enzyme Mix to the flask, and shake the flask at 80 rpm at RT for 1-3 h on a shaker.
  7. Repeatedly pipet the tumor cell suspension to further dissociate cells, centrifuge the cell suspension at 50 x g at RT for 10 min and collect the supernatant. Discard the bigger pellets in the bottom of the tube and centrifuge the supernatant at 200 x g for 5 min.
  8. Wash cell pellets with 10 ml wash buffer at 200 x g for 5 min once.
  9. Suspend the cell pellet with 2 ml ACK lysing buffer for 1 min to deplete red blood cells (observe the cell suspension under a microscope to determine whether red blood cells are depleted completely. If there are still too many red blood cells, add more ACK lysing buffer until there are very few red blood cells).
  10. Centrifuge at 200 x g and remove the supernatant.
  11. Re-suspend cells in 15 ml 40% Percoll and add into a 50 ml tube.
  12. Lay slowly 15 ml 80% Percoll with a syringe and a long needle (#16 gauge) to the bottom of the above 50 ml tube.
  13. Centrifuge at 325 x g at RT for 23 min (ascending rate: 5; descending rate: 5).
  14. Collect the cells at the interface between 40% and 80% Percoll, which should mostly be leukocytes and directly pass the cell suspension through a 40 μm nylon strainer. Wash the strainer with wash buffer and centrifuge the cell suspension at 425 x g at RT for 10 min.
    Note: After centrifugation, approximately 1.5-2.0 x 105 leukocytes could be obtained from a total of 1 x 106 cells from tumor depending on the tumor type. By discontinuous Percoll isolation, tumor cells will be in the bottom layer after centrifugation (Figure 1).
  15. Re-suspend the cells in desired buffer for further experiments, such as phenotyping with flow cytometry and measurement of functions.


    Figure 1. Schematic illustration of the isolation of tumor infiltrating-leukocytes by discontinuous Percoll gradient. Left tube shows that before centrifugation, 80% Percoll is laid under the total cells suspended in 40% Percoll. Right tube shows that after centrifugation, leukocytes are located at the interface between 40% and 80% Percoll, tumor cells are in the bottom of the tube.

Recipes

  1. 10x Triple Enzyme stock solution (100 ml)
    Mix 1 g Collagenase IV, 100 mg Hyaluronidase and 20,000 Units DNase IV into 80 ml HBSS
    Add HBSS to 100 ml
    Filter (0.22 μm) sterilize the stock solution
    Store 5 ml aliquots at -20 °C
    Thaw at RT (NOT 37 °C) before use.
  2. Wash buffer (1,000 ml)
    Mix 1 g BSA and 2 ml 0.5 M EDTA with 800 ml HBSS
    Add HBSS to 1,000 ml
  3. 40% Percoll, 5 ml
    Mix 0.6 ml 10x PBS, 5.4 ml Percoll and 9 ml DMEM
  4. 80% Percoll, 15 ml
    Mix 1.2 ml 10x PBS, 10.8 ml Percoll and 3 ml DMEM

Acknowledgments

This protocol is adapted from Liu et al. (2013).

References

  1. Liu, Y., Chen, K., Wang, C., Gong, W., Yoshimura, T., Liu, M. and Wang, J. M. (2013). Cell surface receptor FPR2 promotes antitumor host defense by limiting M2 polarization of macrophages. Cancer Res 73(2): 550-560.

简介

癌症相关炎症的标志是白细胞浸润到肿瘤中,据信这是由趋化因子募集的。 一些浸润性白细胞如巨噬细胞通常通过产生生长诱导和血管生成因子来促进肿瘤生长。 在这里,我们描述了一种用Percoll密度梯度分离肿瘤浸润白细胞的方法,因为Percoll具有低粘度,低渗透压和对细胞无毒性。 基于它们的密度分离不同的白细胞群体,并在40%和80%不连续的Percoll梯度之间的界面处收集。

材料和试剂

  1. 肿瘤小鼠
  2. 胶原酶,IV型(Sigma-Aldrich,目录号:C5138)
  3. DNase,IV型(Sigma-Aldrich,目录号:D5025)
  4. Fungizone Antimycotic(Life Technologies,Invitrogen TM ,目录号:15290-026)
  5. Hank's平衡盐溶液(HBSS),不含氯化钙或氯化镁(Lonza,目录号:10-547F)
  6. 透明质酸酶,V型(Sigma-Aldrich,目录号:H6254)
  7. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A4503)
  8. 0.5M EDTA(Quality Biological Inc,目录号:qb351-027-721)
  9. Percoll(GE Healthcare,目录号:17-0891-01)
  10. Dulbecco's Modified Eagle's Medium具有稳定的L-谷氨酰胺(DMEM)的高葡萄糖(Lonza,目录号:12-614F)
  11. 胎牛血清(FBS)(Thermo Fisher Scientific,目录号:SH30070-03)
  12. 10x PBS(Life Technologies,Gibco ,目录号:70011)
  13. ACK裂解缓冲液(Lonza,目录号:10-548E)
  14. 10x三重酶储备液(参见配方)
  15. 洗涤缓冲液(见配方)
  16. 40%Percoll(见配方)
  17. 80%Percoll(见配方)

设备

  1. 25ml倾斜颈和蓝色塞密封帽,非通气组织培养烧瓶(BD Biosciences,Falcon ,目录号:353018)
  2. 振动器(Lab-Line,型号:1314)
  3. 离心机
  4. 100 mm培养皿

程序

  1. 生长在小鼠上的解剖Lewis肺癌(LLC)肿瘤(肿瘤体积〜1,000mm 3 /肿瘤,肿瘤重量:0.5〜0.8g /肿瘤)。
  2. 将一个肿瘤放入100mm培养皿中,在室温(RT)下加入5-10ml HBSS
  3. 用手术刀快速切开肿瘤,将碎片小到足以吸入5ml移液管中,而不会被卡在RT。
  4. 将肿瘤组织悬液转移到50ml非通气组织培养瓶中
  5. 用至多40ml HBSS冲洗培养皿,并将悬浮液转移到烧瓶中。 烧瓶中的总体积为45ml
  6. 向烧瓶中加入5ml 10x三重酶混合物,并在室温下在80rpm下在摇床上摇动烧瓶1-3小时。
  7. 重复移液肿瘤细胞悬液进一步离解细胞,在50×g离心细胞悬液在室温10分钟,收集上清液。弃去管底部的较大颗粒,并以200×g离心上清液5分钟。
  8. 用200ml×10ml的洗涤缓冲液洗涤细胞沉淀5分钟,每次5分钟
  9. 用2毫升ACK裂解缓冲液悬浮细胞沉淀1分钟以消耗红细胞(在显微镜下观察细胞悬浮液以确定红细胞是否完全耗尽如果仍然有太多的红细胞,添加更多的ACK裂解缓冲液,直到红细胞很少)。
  10. 以200×g离心,除去上清液
  11. 将细胞重悬在15ml 40%Percoll中,并加入50ml管中
  12. 用注射器和长针(#16规格)将15ml 80%Percoll缓慢放置到上述50ml管的底部。
  13. 在室温下以325×g离心23分钟(上升速率:5;下降速率:5)。
  14. 收集细胞在40%和80%Percoll之间的界面,这应该主要是白细胞和直接通过一个40微米的尼龙过滤器的细胞悬浮液。用洗涤缓冲液洗涤过滤器,并在室温下将细胞悬浮液以425×g离心10分钟。
    注意:离心后,取决于肿瘤类型,可从肿瘤的总共1×10 6个细胞中获得约1.5-2.0×10 5个白细胞。通过不连续的Percoll分离,离心后肿瘤细胞将在底层(图1)。
  15. 将细胞重新悬浮在所需的缓冲液中用于进一步的实验,例如用流式细胞术进行表型和测量功能。

    图1.通过不连续的Percoll梯度分离肿瘤浸润性白细胞的示意图。左管显示在离心之前,将80%Percoll置于悬浮于40%Percoll中的总细胞下。右管显示,离心后,白细胞位于40%和80%之间的界面Percoll,肿瘤细胞在管的底部。

食谱

  1. 10x三重酶储备溶液(100ml) 将1μg胶原酶IV,100mg透明质酸酶和20,000单位DNase IV混合到80ml HBSS中
    将HBSS添加至100 ml
    过滤器(0.22μm)对储备液进行灭菌
    将5毫升等分试样储存在-20℃下
    使用前在室温下(非37°C)解冻。
  2. 洗涤缓冲液(1,000 ml)
    将1μgBSA和2ml 0.5M EDTA与800ml HBSS混合 将HBSS添加至1,000 ml
  3. 40%Percoll,5ml
    混合0.6ml 10x PBS,5.4ml Percoll和9ml DMEM
  4. 80%Percoll,15ml
    混合1.2ml 10x PBS,10.8ml Percoll和3ml DMEM

致谢

该协议改编自Liu et al。(2013)。

参考文献

  1. Liu,Y.,Chen,K.,Wang,C.,Gong,W.,Yoshimura,T.,Liu,M.and Wang,J.M。 细胞表面受体FPR2通过限制巨噬细胞的M2极化来促进抗肿瘤宿主防御。 Cancer Res 73(2):550-560。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Liu, Y., Chen, K., Wang, C., Gong, W., Yoshimura, T., Wang, J. M. and Liu, M. (2013). Isolation of Mouse Tumor-Infiltrating Leukocytes by Percoll Gradient Centrifugation. Bio-protocol 3(17): e892. DOI: 10.21769/BioProtoc.892.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。