搜索

Differentiation of THP1 Cells into Macrophages for Transwell Co-culture Assay with Melanoma Cells
THP1细胞分化成巨噬细胞及与黑色素瘤细胞的跨室联合培养试验   

评审
匿名评审
下载 PDF 引用 收藏 提问与回复 分享您的反馈

本文章节

Abstract

Understanding how immune cells such as macrophages interact with cancer cells is of increasing interest, as cancer treatments move towards combing both targeted- and immuno-therapies in new treatment regimes. This protocol is using THP-1 cells, a human leukemia monocytic cell line that can be differentiated into macrophages. This allows studying the effects of the macrophage secretome on cancer cells (on e.g., growth, drug response or gene expression) in co-cultures without direct cell contact interactions. This is an important aspect as it removes the presence of any phagocytic aspect to changes in the cancer cell number and behaviour. The in vitro THP-1 monocyte differentiation into polarized macrophages was used to study the effects of both M1 and M2 type populations of macrophages on melanoma cells (Smith et al., 2014; Tsuchiya et al., 1980). M1 type macrophages are classically thought to be tumour suppressing as opposed to M2 type macrophages, which are thought to possess tissue repairing and tumour growth promoting activities.

Keywords: Co-culture(共培养), Transwell(Transwell), Macrophages(巨噬细胞), THP1(THP1), Melanoma(黑色素瘤)

Materials and Reagents

  1. Transwell inserts 0.4 μM (BD Biosciences, catalog number: 353493 )
    Note: Currently, it is “Corning, Falcon®, catalog number: 353493 ”.
  2. Sterile 15 ml centrifuge tubes
  3. 20 µl and 1 ml tips
  4. Corning® 6 well cell culture plates (Sigma-Aldrich, catalog number: CLS3516-50EA )
  5. THP-1 cells (ATCC, catalog number: ATCC® TIB-202TM )
  6. Foetal bovine serum (500 ml) (heat inactivated by manufacture) (Thermo Fisher Scientific, GibcoTM, catalog number: 10500064 )
  7. Penicillin/Streptomycin soln (100x) stabilised (100 ml) (Sigma-Aldrich, catalog number: P4333 )
  8. Dulbeccos PBS (1x) w/o Ca2+ or Mg2+ (500 ml) (Sigma-Aldrich, catalog number: PBS2541 )
  9. Trypsin-EDTA (1x) (100 ml) (Sigma-Aldrich, catalog number: T3924-100 ml )
  10. EDTA disodium salt AR E (500 gm) (Thermo Fisher Scientific, Fisher Scientific, catalog number: FKCF/0700/53 )
  11. DMEM w/L-Glutamine, Pyruvate & sodium bicarbonate (500 ml) (Sigma-Aldrich, catalog number: D6429-24x )
  12. RPMI 1640 w/L-Glutamine-Bicarbonate (500 ml) (Sigma-Aldrich, catalog number: R8758 )
  13. 12-O-tetradecanoylphorbol-l3-acetate (PMA) (Sigma-Aldrich, catalog number: P1585 )
  14. Recombinant human interferon type gamma (IFN-γ) (Peprotech, catalog number: 300-02 )
  15. Recombinant human interleukin 4 (IL-4) (Peprotech, catalog number: 200-04 )
  16. Recombinant human interleukin 13 (IL-13) (Peprotech, catalog number: 200-13 )
  17. Lipopolysaccharides from Escherichia coli (Sigma-Aldrich, catalog number: L2630 )
  18. DMEM/FCS media (see Recipes)
  19. RPMI/FCS media (see Recipes)
  20. IL4 stock solution (see Recipes)
  21. IL13 stock solution (see Recipes)
  22. IFN-γ stock solution (see Recipes)
  23. PMA (see Recipes)
  24. LPS (see Recipes)

Equipment

  1. Benchtop Centrifuge
  2. Aspirator
  3. Haemocytometer
  4. Tissue culture hood
  5. CO2 incubator

Procedure

Schematic of the protocol
Below is a diagram showing a brief outline of the steps of the protocol and a timeline indicating the order of events all carried out under sterile conditions.


Figure 1. A schematic diagram of the procedure. THP-1 cells (yellow) are placed into the insert, where they are activated by PMA; addition of LPS induces M1 macrophage (M1 MΦ), IL4/IL13 induces M2 MΦ differentiation (green cells).


Day 1
THP-1 cells are grown in suspension in RPMI/FCS media, and activation is performed once plated into transwell inserts. Inserts are handled at the sterile edges of the inserts using gloves.

  1. THP-1 cells are counted using a haemocytometer a cell suspension with a concentration of 1 x 105 cells/ml is made up.
  2. One 6 well-plate is then prepared by adding 2 ml of RPMI/FCS media per 6 well. The transwell inserts are then placed into the 6 well containing the 2 ml medium.
  3. 2 ml of the THP-1 cell suspension is added into the inserts (2 x 105 cells/insert).
  4. Once settled THP-1 cells are treated immediately with 10 ng/ml of 12-O-tetradecanoylphorbol-l3-acetate (PMA) for 24 h (1 µl of stock solution into 4 ml total volume) as described in Figure 1 Day 1.

Day 2
Activated THP-1 cells are differentiated in transwell inserts after 24 h in PMA.

  1. A new 6 well plate is prepared by adding 2 ml of fresh RPMI/FCS media.
  2. The PMA containing media is then aspirated gently from the inserts containing the activated THP-1 cells and the inserts are transferred to the new 6 well plate as described in Figure 1 Day 2.
  3. 2 ml of RPMI/FCS media is then added into the inserts and left for 1 h before treatment.
  4. The inserts are then treated with the desired mix of cytokines:
    1. M1 differentiation can be achieved either by treating with 15 ng/ml of lipopolysaccharide (LPS) (1.5 µl of stock solution into 4ml) or 50 ng/ml IFN-γ (2 µl of stock solution into 4 ml).
    2. M2 differentiation can be achieved by the combined treatment with 25 ng/ml IL4 and 25 ng/ml IL13 (1 µl of stock solution into 4 ml).
    3. Undifferentiated but activated THP-1 cells are left untreated in the insert to be used as a reference well for activated but non-differentiated monocytes.
    4. Correct differentiation into M1 and M2 type macrophages can be assessed at this stage by immunofluorescence, ELISA or gene expression analysis for specific markers (such as IL1, IL10, ARGINASE) as described in Smith et al. (2014).

Day 3
The human melanoma cell line to be investigated is pre-plated into 6 well plates. Melanoma cells are adherent and are usually grown in DMEM/FCS media. For example WM266-4 cells are plated at 2 x 105/insert for a 72 h co-culture experiment.

  1. Melanoma cells are washed once in 5 ml of PBS/EDTA.
  2. After PBS/EDTA aspiration 1 ml of trypsin/EDTA is added to the flask and then incubated in a 37 °C incubator for 3-5 min.
  3. Cells are then re-suspended in 10 ml of DMEM or RPMI (depending on cell line) media and then counted.
  4. Depending on the doubling rate of cell line used and the length of the experiment being performed the number of cells plated will vary but usually lie within the region of 0.5-2 x 105 cells per 6 well (2 ml per well).

Day 4
Setting up the co-culture by combining the differentiated macrophages and melanoma cells in the same plate as described in Figure 1 Day 4.

  1. 48 h after the addition of LPS or IL4/IL23, the differentiated THP-1cells are washed 3 times in fresh RPMI/FCS media. One wash consisting of careful aspiration of the media and then adding 4 ml of fresh RPMI/FCS media.
  2. The inserts are then picked up using gloves and transferred onto the top of the melanoma cell culture with the addition of 2 ml of DMEM or RPMI (depending on the melanoma cell line) media into the inserts.
    Note: Once transferred, the transwell assay is then ready to be used in several in vitro assays such as drug treatment survival assays or gene expression analysis after defined times of co-culture (Smith et al., 2014). Cells can be kept in this co-culture situation for a further 96 h if desired. This co-culture technique allows for the isolation and study of both populations of cells after the experiment. RNA or protein lysates can be obtained from the adherent melanoma cells at the bottom of the wells, in addition the macrophages can be lysed separately by isolating the insert.

Representative data


Figure 2. Cell number assessment of melanoma cells co-cultured with differentiated macrophages. Crystal violet staining of WM266-4 melanoma cells, which had been co-cultured with M1 and M2 macrophages for 5 days. After the removal of the insert, cells were fixed with 4% formaldehyde/PBS (15 min), stained with 0.5% crystal violet/PBS (1 h) then washed with water until wash-water was clear. Crystal violet very efficiently stains cells and the cell/colony number can assessed by counting or optical density (OD540) measurement after solubilisation of the dye. This step does not require sterile conditions.

Notes

The most crucial factor in producing reliable and consistent results using this transwell technique is choosing the correct number of the respective melanoma cell line for plating so that they do not over-grow by the end-point of the experiment. There is also an increased risk of contamination due to the handling of the inserts above the wells, which can be reduced by working as swift and clean as possible.

Recipes

  1. DMEM/FCS media
    Dulbecco's modified eagle medium supplemented with 1% of Penicillin-streptomycin solution and 10% FCS
  2. RPMI/FCS media
    RPMI supplemented with 1% of Penicillin-streptomycin solution and 10% FCS
  3. IL4 stock solution
    Made up to 100 µg/µl in sterile PBS 0.05% BSA and aliquoted and stored at -80 °C
  4. IL13 stock solution
    Made up to 100 µg/µl in sterile PBS 0.05% BSA and aliquoted and stored at -80 °C
  5. IFN-γ stock solution
    Made up to 100 µg/µl in sterile PBS 0.05%BSA and aliquoted and stored at -80 °C
  6. PMA
    Diluted in sterile PBS to concentrations of 40 µg/µl and then aliquoted and stored at -20 °C
  7. LPS
    Diluted in sterile PBS to concentrations of 40 µg/µl and then aliquoted and stored at -20 °C

Acknowledgments

The THP-1 differentiation protocol and development of this system was done with the guidance of Nadia Lunheshi and Richard Sainson at Medimmune Ltd, UK. Funding for this research was provided by CRUK C11591/A16416 awarded to C. Wellbrock. This protocol was developed and used in Smith et al. (2014).

References

  1. Smith, M. P., Sanchez-Laorden, B., O'Brien, K., Brunton, H., Ferguson, J., Young, H., Dhomen, N., Flaherty, K. T., Frederick, D. T., Cooper, Z. A., Wargo, J. A., Marais, R. and Wellbrock, C. (2014). The immune microenvironment confers resistance to MAPK pathway inhibitors through macrophage-derived TNFalpha. Cancer Discov 4(10): 1214-1229.
  2. Tsuchiya, S., Yamabe, M., Yamaguchi, Y., Kobayashi, Y., Konno, T. and Tada, K. (1980). Establishment and characterization of a human acute monocytic leukemia cell line (THP-1). Int J Cancer 26(2): 171-176.

简介

了解免疫细胞如巨噬细胞如何与癌细胞相互作用越来越感兴趣,因为癌症治疗趋向于在新的治疗方案中结合靶向和免疫治疗。该协议使用THP-1细胞,人类白血病单核细胞系,可以分化成巨噬细胞。这允许在没有直接细胞接触相互作用的共培养物中研究巨噬细胞分泌蛋白组织对癌细胞的作用(例如生长,药物反应或基因表达)。这是一个重要的方面,因为它消除了任何吞噬方面的癌细胞数量和行为的变化的存在。将体外THP-1单核细胞分化为极化巨噬细胞用于研究巨噬细胞的M1和M2型群体对黑素瘤细胞的作用(Smith等人,2014年, ; Tsuchiya等人,1980)。 M1型巨噬细胞通常被认为是与M2型巨噬细胞相反的肿瘤抑制,M2型巨噬细胞被认为具有组织修复和促进肿瘤生长的活性。

关键字:共培养, Transwell, 巨噬细胞, THP1, 黑色素瘤

材料和试剂

  1. Transwell插入0.4μM(BD Biosciences,目录号:353493)
    注意:目前为"Corning,Falcon?,目录号:353493"。
  2. 无菌15 ml离心管
  3. 20μl和1 ml提示
  4. 6孔细胞培养板(Sigma-Aldrich,目录号:CLS3516-50EA)中。
  5. THP-1细胞(ATCC,目录号:ATCC TIB-202 TM
  6. 胎牛血清(500ml)(通过制造热灭活)(Thermo Fisher Scientific,Gibco TM ,目录号:10500064)
  7. 青霉素/链霉素溶液(100x)稳定(100ml)(Sigma-Aldrich,目录号:P4333)
  8. Dulbeccos PBS(1x)w/o Ca 2+或Mg 2+(500ml)(Sigma-Aldrich,目录号:PBS2541)
  9. 胰蛋白酶-EDTA(1x)(100ml)(Sigma-Aldrich,目录号:T3924-100ml)
  10. EDTA二钠盐ARE(500gm)(Thermo Fisher Scientific,Fisher Scientific,目录号:FKCF/0700/53)
  11. DMEM w/L-谷氨酰胺,丙酮酸&碳酸氢钠(500ml)(Sigma-Aldrich,目录号:D6429-24x)
  12. RPMI 1640w/L-谷氨酰胺 - 碳酸氢盐(500ml)(Sigma-Aldrich,目录号:R8758)
  13. 12-O-十四酰基佛波醇-13-乙酸酯(PMA)(Sigma-Aldrich,目录号:P1585)
  14. 重组人干扰素型γ(IFN-γ)(Peprotech,目录号:300-02)
  15. 重组人白介素4(IL-4)(Peprotech,目录号:200-04)
  16. 重组人白细胞介素13(IL-13)(Peprotech,目录号:200-13)
  17. 来自大肠杆菌的脂多糖(Sigma-Aldrich,目录号:L2630)
  18. DMEM/FCS介质(参见配方)
  19. RPMI/FCS介质(参见配方)
  20. IL4储备溶液(见配方)
  21. IL13储备溶液(见配方)
  22. IFN-γ储备溶液(见配方)
  23. PMA(参见配方)
  24. LPS(见配方)

设备

  1. 台式离心机
  2. 吸气器
  3. 血细胞计数器
  4. 组织培养罩
  5. CO <2>孵化器

程序

协议示意图
下面是显示协议步骤的简要概述和指示所有在无菌条件下进行的事件顺序的时间线。


图1.程序的示意图。将THP-1细胞(黄色)置于插入物中,在那里它们被PMA激活;加入LPS诱导M1巨噬细胞(M1MΦ),IL4/IL13诱导M2MΦ分化(绿细胞)。


第1天
THP-1细胞在RPMI/FCS培养基中的悬浮液中生长,并且一旦接种到transwell插入物中就进行激活。使用手套在插入件的无菌边缘处处理插入件。

  1. 使用血细胞计数器计数THP-1细胞,制备浓度为1×10 5个细胞/ml的细胞悬液。
  2. 然后通过加入2ml RPMI/FCS培养基制备一个6孔板 每6孔。然后将transwell插入物放入6孔中 含有2ml培养基。
  3. 将2ml的THP-1细胞悬浮液加入到插入物中(2×10 5个细胞/插入物)。
  4. 一旦沉降的THP-1细胞立即用10ng/ml的 12-O-十四烷酰佛波醇-13-乙酸酯(PMA)处理24小时(1μl原液 溶液加入4ml总体积),如图1第1天所述。

第2天
在PMA中24小时后,在transwell插入物中分化活化的THP-1细胞。

  1. 通过加入2ml新鲜的RPMI/FCS培养基制备新的6孔板
  2. 然后从插入物中轻轻地吸出含有PMA的培养基 含有活化的THP-1细胞并转移插入片段 新的6孔板如图1第2天所述。
  3. 然后将2ml RPMI/FCS培养基加入到插入物中,并在处理前放置1小时
  4. 然后用所需的细胞因子混合物处理插入物:
    1. M1分化可以通过用15ng/ml处理来实现 的脂多糖(LPS)(1.5μl储备液加入4ml)或50μl ng/ml IFN-γ(2μl储备液加入4ml)
    2. M2 可以通过用25ng/ml的联合治疗实现分化 IL4和25ng/ml IL13(1μl储备液加入4ml)中
    3. 未分化但活化的THP-1细胞未经处理 插入用作激活的参考孔,但是 非分化的单核细胞。
    4. 正确分化为M1 和M2型巨噬细胞可以在这个阶段评估 免疫荧光,ELISA或基因表达分析 标记(如IL1,IL10,ARGINASE),如Smith等人(2014)中所述。

第3天
将待研究的人黑素瘤细胞系预铺板到6孔板中。黑素瘤细胞是粘附的,并且通常在DMEM/FCS培养基中生长。例如,WM266-4细胞以2×10 5个/插入物接种72小时共培养实验。

  1. 黑色素瘤细胞在5ml PBS/EDTA中洗涤一次
  2. 在PBS/EDTA吸出后,向烧瓶中加入1ml胰蛋白酶/EDTA,然后在37℃培养箱中温育3-5分钟。
  3. 然后将细胞重悬于10ml DMEM或RPMI(取决于细胞系)培养基中,然后计数
  4. 取决于使用的细胞系的倍增速率和长度 进行的实验中,铺板的细胞数目将变化 通常位于每6孔0.5-2×10 5个细胞的区域内(2ml / 好)。

第4天
通过在如图1所述的相同板中组合分化的巨噬细胞和黑素瘤细胞来建立共培养物第4天。

  1. 48小时后加入LPS或IL4/IL23,分化 将THP-1细胞在新鲜RPMI/FCS培养基中洗涤3次。一次洗 包括小心抽吸的媒体,然后加入4毫升 新鲜RPMI/FCS培养基
  2. 然后使用手套拿起插入物 ?并转移到黑色素瘤细胞培养物的顶部 加入2ml DMEM或RPMI(取决于黑素瘤细胞系) 媒体插入插页 注意:一旦转移,transwell测定 然后准备用于几种体外测定例如药物 治疗后存活分析或基因表达分析 共培养时间(Smith等,2014)。细胞可以保存在这里 共培养情况下再培养96小时。这种共同文化 技术允许隔离和研究两种群体 细胞。 RNA或蛋白质裂解物可以从中获得 粘附黑素瘤细胞在孔底,另外 巨噬细胞可以通过分离插入物单独裂解。

代表数据


图2.与分化的巨噬细胞共培养的黑素瘤细胞的细胞数量评估。已经与M1和M2巨噬细胞共培养5天的WM266-4黑素瘤细胞的结晶紫染色。在移除插入物之后,用4%甲醛/PBS(15分钟)固定细胞,用0.5%结晶紫/PBS(1小时)染色,然后用水洗涤直至洗涤水澄清。结晶紫非常有效地染色细胞,并且可以通过在染料溶解后计数或光密度(OD540)测量来评估细胞/集落数。该步骤不需要无菌条件。

笔记

使用该transwell技术产生可靠且一致的结果的最关键的因素是选择用于电镀的各个黑素瘤细胞系的正确数目,使得它们在实验的终点不过度生长。由于在井上方处理插入物也存在增加的污染风险,这可以通过尽可能迅速和清洁地工作来减少。

食谱

  1. DMEM/FCS介质
    补充有1%青霉素 - 链霉素溶液和10%FCS的Dulbecco改良的Eagle培养基
  2. RPMI/FCS媒体
    补充有1%青霉素 - 链霉素溶液和10%FCS的RPMI
  3. IL4储备液
    在无菌PBS 0.05%BSA中补加至100μg/μl,并等分并保存在-80℃下
  4. IL13储备液
    在无菌PBS 0.05%BSA中补加至100μg/μl,并等分并保存在-80℃下
  5. IFN-γ储备液
    在无菌PBS 0.05%BSA中补加至100μg/μl,并等分并保存在-80℃下
  6. PMA
    在无菌PBS中稀释至浓度为40μg/μl,然后等分并储存在-20℃下
  7. LPS
    在无菌PBS中稀释至浓度为40μg/μl,然后等分并储存在-20℃下

致谢

THP-1分化方案和该系统的开发是在Nadia Lunheshi和Richard Sainson在Medimmune Ltd,UK的指导下进行的。该研究的资金由授予C.Weborbrock的CRUK C11591/A16416提供。该协议在Smith等人(2014)中开发和使用。

参考文献

  1. Smith,MP,Sanchez-Laorden,B.,O'Brien,K.,Brunton,H.,Ferguson,J.,Young,H.,Dhomen,N.,Flaherty,KT,Frederick,DT,Cooper,ZA, Wargo,JA,Marais,R.and Wellbrock,C。(2014)。 免疫微环境通过巨噬细胞衍生的TNFα赋予对MAPK通路抑制剂的抗性。 Cancer Discov 4(10):1214-1229。
  2. Tsuchiya,S.,Yamabe,M.,Yamaguchi,Y.,Kobayashi,Y.,Konno,T.and Tada,K。(1980)。 人类急性单核细胞白血病细胞系(THP-1)的建立和表征。 Int J Cancer 26(2):171-176
  • 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: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Smith, M. P., Young, H., Hurlstone, A. and Wellbrock, C. (2015). Differentiation of THP1 Cells into Macrophages for Transwell Co-culture Assay with Melanoma Cells. Bio-protocol 5(21): e1638. DOI: 10.21769/BioProtoc.1638.
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。