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Mouse Embryonic Fibroblast Cell Culture and Stimulation
小鼠胚胎成纤维细胞的培养和免疫刺激   

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Abstract

Culture of mouse embryonic fibroblast (MEF) cells represents a powerful system to test gene function due to their easy accessibility, rapid growth rates, and the possibility of a large number of experiments. Fibroblasts are a group of heterogeneous resident cells of mesenchymal origin that have various locations, diverse appearances and distinctive activities. Because of their ubiquitous distribution as tissue cells, these cells are poised to respond to factors released by newly activated innate immune cells, thus becoming a useful tool to study inflammation and immunity. Here, we describe procedures for mouse embryonic fibroblast cell isolation, primary culture, and stimulation. Specifically, we have optimized a step of serum starvation prior to stimulation. This step is necessary to maintain the quiescent status of these cells before they are exposed to pro-inflammatory stimuli for optimal responses. As shown in our previous studies, these mouse fibroblasts do not express Tnf, Csf2 or Il2 mRNAs at levels readily detectable by routine northern blotting techniques (Lai WS et al., 2006).

Keywords: Mouse embryonic fibroblast(小鼠胚胎成纤维细胞), Primary cell culture(初级细胞培养), Tumor necrosis factor(肿瘤坏死因子), Serum starvation(血清饥饿)

Materials and Reagents

  1. 60-mm sterile petri dish (e.g., BD Biosciences, Falcon®, catalog number: 353002 )
    Note: Currently, it is “Corning, Falcon®, catalog number: 353002”.
  2. 100-mm sterile petri dish (e.g., Corning, catalog number: 430167 )
    Note: Currently, it is “Corning, Falcon®, catalog number: 430167”.
  3. 50 ml sterile conical tube (e.g., BD Biosciences, Falcon®, catalog number: 352070 )
    Note: Currently, it is “Corning, Falcon®, catalog number: 352070”.
  4. 5 ml sterile serological pipettes (e.g., Corning Costar®, catalog number: 4051 )
  5. Mice (Male and female, ideally at 8-12 weeks of age)
  6. Ice
  7. Recombinant mouse tumor necrosis factor (TNF) (R&D Systems, catalog number: 410-MT )
  8. 1x Phosphate-buffered saline (PBS) without calcium and magnesium
  9. 70% ethanol
  10. 0.25% trypsin/EDTA (Thermo Fisher Scientific, GibcoTM, catalog number: 25200-056 )
  11. 0.05% trypsin/EDTA (Thermo Fisher Scientific, GibcoTM, catalog number: 25300 )
  12. Fetal bovine serum defined (FBS) (GE Healthcare, HyClone, catalog number: SH30070.03 )
  13. Dulbecco’s modified Eagle medium (DMEM) (Thermo Fisher Scientific, GibcoTM, catalog number: 11965-092 )
  14. Penicillin-Streptomycin 10,000 U/ml (Thermo Fisher Scientific, GibcoTM, catalog number: 15140-122 )
  15. L-glutamine 200 mM (Thermo Fisher Scientific, GibcoTM, catalog number: 25030-081 )
  16. Rinsing medium (see Recipes)
  17. Complete medium (see Recipes)
  18. Serum-starving medium (see Recipes)

Equipment

  1. Dissecting microscope (e.g., Leica Microsystems, model: MZ6 )
  2. Dissecting instruments: tweezers and scissors
  3. NuAire Biological Safety Cabinet, class II, or similar
  4. 37 °C, 5% CO2 forced-air incubator (e.g., Thermo Fisher Scientific, FormaTM, model: 3110 )
  5. Centrifuge with swinging-bucket rotor and adaptors for 50-ml conical tubes
  6. CO2 chamber for mouse euthanization
  7. 37 °C shaker water bath (e.g., Thermo Fisher Scientific, PrecisionTM, model: Shaker Bath 25 )
  8. Autoclaved razor blades

Procedures

  1. Set up timed-matings between male and female mice
    1. Check for the plugs on the following day. The presence of a vaginal plug is considered embryonic day 0.5 (E0.5).


      Figure 1. The presence of a vaginal plug is identified (arrowhead) in a female mouse on the second day following timed-mating

    2. Monitor weight gain in female mice every 2-3 days.
    3. Pregnant female mice of gestational days 14.5-15.5 are used.
      Notes:
      1. Pregnancy in female mice is usually accompanied by weight gain over time, as well as a visibly round belly.
      2. The weight gain for pregnant female mice at gestational day 15.5 ranges between 7.5 and 15.6 g, with an average weight gain of 11.5 g.

  2. Embryo removal and cell disaggregation
    1. Euthanize a pregnant female mouse at E14.5 or 15.5, swab abdomen with 70% ethanol, cut open the abdomen and remove the uterus, placing it in a 10-cm petri dish containing 10 ml sterile PBS on ice (Figure 2A).
    2. Cut away the uterine decidua, separate and transfer each embryo along with its yolk sac to a fresh 60-mm petri dish with 5 ml PBS on ice (Figure 2B-C).
    3. Carefully remove the yolk sac and placenta under a dissecting microscope, and examine the embryo to determine viability and/or morphological changes (Figure 2D).
      Note: We routinely check for embryo size, color (pale, hemorrhage, etc.), and any obvious structural alterations including eye development and limb formation, as well as neurological abnormalities, such as spinal bifida and anencephaly. These are the two most common presentations in our experience, although both are rare in wild-type mice.
    4. Cut the tail using clean scissors if DNA for genotyping is desired.
      Note: Clean the instruments with 70% ethanol between embryos at this step to avoid carryover contamination.
    5. Decapitate the head from the body with a pair of clean tweezers, lay the embryo on its back, and remove the visible internal organs (Figure 2D-E).
    6. Put the body in another sterile 60-mm petri dish containing 0.5 ml rinsing medium on ice, and move them to a tissue culture hood (Figure 2F).
    7. Mince the bodies with fresh, sterile razor blades into small fragments as fine as possible (Figure 2G).
    8. Add 5 ml of cell culture-grade Trypsin-EDTA (0.25%), and transfer to a 50 ml conical tube after pipetting up and down several times with a 5 ml pipette to disaggregate the tissue.
    9. Place the tube in a 37 °C water bath with shaking for enzymatic digestion for 1-2 h.
    10. Add another 5 ml of rinsing medium, vortex and spin at 1,200 x g for 5 min at room temperature.


      Figure 2. Procedures for the isolation of MEFs. C-F, images were captured under a dissecting microscope. PL, placenta, YS, yolk sac, UC, umbilical cord, IO, internal organs.

  3. Culture and passage of mouse embryonic fibroblasts 
    1. To obtain cells for culture, carefully place a 5 ml pipette through the supernatant and pipette up the pellet along with 0.5-1 ml of the supernatant (Figure 2H, 2I), and place in a 100-mm dish containing 10 ml complete culture medium for each embryo. Pipet cells up and down in the culture medium to disperse the cell clumps. Allow cells to adhere and grow in a 37 °C incubator supplied with 5% CO2. This is passage 0.
      Note: Don’t try to aspirate the supernatant using a vacuum, since the DNA released from the cells makes it difficult to remove the supernatant without removing the cell pellets. The fibroblasts should start to attach to the bottom of dishes within 2 h after plating.
    2. Change the medium the following day.
    3. Monitor cell growth. Once the cells reach confluence (~ 48 h), passage the cells at a dilution of 1:4 into 4 new 100-mm Petri dishes after washing with 10 ml of PBS, trypsinization with 2 ml of 0.05% trypsin/EDTA, and neutralization with 8 ml of complete culture medium. This is passage 1.
    4. Continue to expand cells when the cells are near confluence (usually within 48 h). Passage cells with trypsin treatment as above and re-plate cells into another four 100-mm Petri dishes. This is passage 2.
      Note: Cells can be passaged at 1:3 to 1:5 depending on cell growth rates. The maximum number of passages for healthy primary MEFs is approximately 4, as cell growth is slowed beyond passages 4-5. We normally work with MEFs at passages 2-4 to get optimal cellular responses to stimuli.

  4. Stimulation of MEFs and cell harvest
    1. Confluent MEFs at passages 1-3 in 100-mm Petri dishes are collected and re-plated into 60-mm Petri dishes at an initial seeding density of 2-3 x 105 cells in 5 ml culture medium per petri dish. These cells are used at passages 2-4.
      Note: The estimate for the cell number in an almost confluent 100-mm petri dish is about 10-20 x 106 cells.
    2. When the cells reach 70-80% confluence, the medium is replaced with 4.5 or 5 ml serum-starving medium for at least 16 h after one wash in serum-free DMEM.
    3. Add 0.5 ml serum or recombinant murine TNF into the serum-starving medium for final concentrations of 10% serum or 1-10 ng/ml of TNF as stimuli.
    4. Cells are then harvested for downstream applications after treatment at the indicated time points. For RNA extraction, cells are lysed with extraction buffer in dishes after one wash with ice-cold PBS. Representative data are shown in Figure 1.

Representative data



Figure 1. Time courses of induction of TNF-responsive genes in mouse fibroblasts. Expression of Fos and Cxcl1 mRNAs in serum-deprived MEFs from wild-type (WT) and tristetraprolin (TTP)-deficient (KO) mice was examined at various time points after addition of TNF at 10 ng/ml, using real-time RT-PCR. Results shown are means ± SEM of five independent experiments, and data are expressed as fold changes relative to WT at time 0; this value was set as one after normalizing to an internal control, Actb mRNA. Statistical differences between the WT and KO means, determined by paired Student t tests, are indicated (*P < 0.05, WT vs. KO). Modified from Qiu et al. (2015) with permission.

Recipes

  1. Rinsing medium
    DMEM
    1% Penicillin-Streptomycin
  2. Complete medium
    DMEM
    10% FBS
    1% Penicillin-Streptomycin
    2 mM Glutamine
  3. Serum-starving medium
    DMEM
    0.5% FBS
    1% Penicillin-Streptomycin
    2 mM Glutamine

Acknowledgments

This protocol was adapted from previously published studies, Lai et al. (2006) and Horner et al. (2009), and was used in Qiu et al. (2015). We thank Dee Wenzel for husbandry support, Christopher McGee for assistance with live animal imaging, and Drs. Melissa Wells and Diana Cruz-Topete for comments on the protocol. This research was supported by the Intramural Research Program of the National Institute of Environmental Health Sciences, National Institutes of Health.

References

  1. Horner, T. J., Lai, W. S., Stumpo, D. J. and Blackshear, P. J. (2009). Stimulation of polo-like kinase 3 mRNA decay by tristetraprolin. Mol Cell Biol 29(8): 1999-2010.
  2. Lai, W. S., Parker, J. S., Grissom, S. F., Stumpo, D. J. and Blackshear, P. J. (2006). Novel mRNA targets for tristetraprolin (TTP) identified by global analysis of stabilized transcripts in TTP-deficient fibroblasts. Mol Cell Biol 26(24): 9196-9208.
  3. Qiu, L. Q., Lai, W. S., Bradbury, A., Zeldin, D. C. and Blackshear, P. J. (2015). Tristetraprolin (TTP) coordinately regulates primary and secondary cellular responses to proinflammatory stimuli. J Leukoc Biol 97(4): 723-736.

简介

小鼠胚胎成纤维细胞(MEF)细胞的培养代表了测试基因功能的强大系统,因为它们容易获得,快速生长速率和大量实验的可能性。成纤维细胞是一组具有不同位置,不同外观和独特活动的间充质来源的异质驻留细胞。由于它们作为组织细胞的普遍分布,这些细胞准备响应由新激活的先天免疫细胞释放的因子,因此成为研究炎症和免疫的有用工具。在这里,我们描述了小鼠胚胎成纤维细胞分离,原代培养和刺激的程序。具体来说,我们已经优化了刺激前血清饥饿的步骤。这个步骤是必要的,以维持这些细胞在它们暴露于促炎刺激为最佳反应之前的静止状态。如在我们以前的研究中所示,这些小鼠成纤维细胞在通过常规Northern印迹技术容易检测的水平下不表达Tnf , Csf2 或 Lai WS等人,2006)。

关键字:小鼠胚胎成纤维细胞, 初级细胞培养, 肿瘤坏死因子, 血清饥饿

材料和试剂

  1. 60-mm无菌培养皿(例如,BD Biosciences,Falcon ,目录号:353002)。
    注意:目前,"Corning,Falcon ? ,目录号:353002"。
  2. 100-mm无菌培养皿(例如,Corning,目录号:430167)
    注意:目前,"Corning,Falcon ? ,目录号:430167"
  3. 50ml无菌锥形管(例如BD Biosciences,Falcon ,目录号:352070)。
    注意:目前,"Corning,Falcon ? ,目录号:352070"
  4. 5ml无菌血清移液管(例如,,Corning Costar ,目录号:4051)
  5. 小鼠(雄性和雌性,理想地在8-12周龄)

  6. 重组小鼠肿瘤坏死因子(TNF)(R& D Systems,目录号:410-MT)
  7. 1x不含钙和镁的磷酸盐缓冲盐水(PBS)
  8. 70%乙醇
  9. 0.25%胰蛋白酶/EDTA(Thermo Fisher Scientific,Gibco< sup> TM,目录号:25200-056)
  10. 0.05%胰蛋白酶/EDTA(Thermo Fisher Scientific,Gibco TM ,目录号:25300)
  11. 定义的胎牛血清(FBS)(GE Healthcare,HyClone,目录号:SH30070.03)
  12. Dulbecco改良的Eagle培养基(DMEM)(Thermo Fisher Scientific,Gibco TM ,目录号:11965-092)
  13. 青霉素 - 链霉素10,000U/ml(Thermo Fisher Scientific,Gibco< sup>,目录号:15140-122)
  14. L-谷氨酰胺200mM(Thermo Fisher Scientific,Gibco TM ,目录号:25030-081)
  15. 冲洗介质(见配方)
  16. 完整介质(见配方)
  17. 血清饥饿培养基(见配方)

设备

  1. 解剖显微镜(例如,徕卡显微系统公司,型号:MZ6)
  2. 解剖器械:镊子和剪刀
  3. NuAire生物安全柜,II级或类似的
  4. 37℃,5%CO 2强制空气培养箱(例如Thermo Fisher Scientific,Forma TM sup),型号:3110) >
  5. 带有摆动斗式转子的离心机和适用于50 ml锥形管的适配器
  6. CO sub 2室用于小鼠安乐死
  7. 37℃摇床水浴(例如Thermo Fisher Scientific,Precision TM ,型号:Shaker Bath 25)
  8. 高压灭菌剃刀刀片

程序

  1. 设置男性和女性小鼠之间的定时交配
    1. 检查第二天的插头。阴道塞的存在被认为是胚胎第0.5天(E0.5)。


      图1.在定时交配后的第二天,在雌性小鼠中识别阴道插头的存在(箭头)

    2. 每2-3天监测雌性小鼠的体重增加。
    3. 使用孕龄14.5-15.5的怀孕雌性小鼠 注意:
      1. 雌性小鼠的妊娠通常伴随着时间的体重增加,以及明显圆形的腹部。
      2. 怀孕雌性小鼠在妊娠15.5天的体重增加范围在7.5和15.6g之间,平均体重增加11.5g。

  2. 胚胎去除和细胞分解
    1. 在E14.5或15.5处对怀孕的雌性小鼠安乐死,用70%乙醇擦拭腹部,切开腹部并取出子宫,将其置于含有10ml无菌PBS的10cm培养皿中(图2A)。 br />
    2. 切除子宫蜕膜,分离并将每个胚胎连同其卵黄囊一起转移到冰上用5ml PBS的新鲜60-mm培养皿中(图2B-C)。
    3. 在解剖显微镜下小心地取出卵黄囊和胎盘,检查胚胎以确定活力和/或形态变化(图2D)。
      注意:我们常规检查胚胎大小,颜色(苍白,出血等),以及任何明显的结构改变,包括眼睛发育和四肢形成,以及神经系统异常,如脊柱裂和无脑畸形。这些是我们的经验中最常见的两种表现,虽然在野生型小鼠中都是罕见的。
    4. 如果需要用于基因分型的DNA,用干净的剪刀剪尾 注意:在此步骤中,在胚胎之间用70%乙醇清洁仪器,以避免残留污染
    5. 用一对干净的镊子将头部从身体上摘下,将胚胎放在其背部,去除可见的内脏(图2D-E)。
    6. 将身体放在另一个无菌的60毫米培养皿中含有0.5毫升冲洗介质在冰上,并移动到组织培养罩(图2F)。
    7. 用新鲜的无菌剃刀刀片将身体切碎成尽可能细的碎片(图2G)。
    8. 加入5毫升细胞培养级胰蛋白酶-EDTA(0.25%),转移到一个50ml锥形管,用移液管上下移动几次后用5ml移液管分解组织。
    9. 将管放置在37℃水浴,摇动酶消化1-2小时
    10. 加入另外5ml的冲洗介质,涡旋,并在室温下以1,200×g旋转5分钟。


      图2.用于分离MEF的程序。C-F,在解剖显微镜下捕获图像。 PL,胎盘,YS,卵黄囊,UC,脐带,IO,内脏
  3. 小鼠胚胎成纤维细胞的培养和传代
    1. 为了获得用于培养的细胞,小心地将5ml吸管通过上清液,用0.5-1ml上清液吸取沉淀物(图2H,2I),并置于含有10ml完全培养基的100-mm培养皿中为每个胚胎。在培养基中上下吸取细胞以分散细胞团块。允许细胞粘附并在提供有5%CO 2的37℃培养箱中生长。这是段落0.
      注意:不要尝试使用真空吸出上清液,因为从细胞释放的DNA使得难以除去上清液而不除去细胞沉淀。成纤维细胞应在铺板后2小时内开始附着在培养皿底部。
    2. 第二天更换介质。
    3. 监测细胞生长。一旦细胞达到汇合(约48小时),将细胞以1:4的稀释度传递到4个新的100-mm培养皿中,用10ml PBS洗涤,用2ml的0.05%胰蛋白酶/EDTA胰蛋白酶化,并中和用8ml完全培养基。这是段1.
    4. 当细胞接近汇合时(通常在48小时内)继续扩增细胞。用上述胰蛋白酶处理的传代细胞并将细胞重新平板成另外四个100mm培养皿。这是段落2.
      注意:细胞可以以1:3至1:5传代,这取决于细胞生长速率。健康初级MEF的最大传代次数约为4,因为细胞生长减缓超过传代4-5。我们通常与MEF在2-4通道工作,以获得最佳的细胞对刺激的反应。

  4. MEF和细胞收获的刺激
    1. 收集在100-mm培养皿中1-3代的汇合MEF,并以5ml培养基中2-3×10 5个细胞的初始接种密度重新接种在60-mm培养皿中每培养皿。这些细胞用于2-4代 注意:几乎融合的100-mm培养皿中细胞数的估计值为约10-20×10 10个 6 个细胞。
    2. 当细胞达到70-80%汇合时,在无血清DMEM中一次洗涤后,用4.5或5ml血清饥饿培养基更换培养基至少16小时。
    3. 向血清饥饿培养基中加入0.5ml血清或重组鼠TNF,最终浓度为10%血清或1-10ng/ml TNF作为刺激物。
    4. 然后在指定的时间点处理后收获细胞用于下游应用。对于RNA提取,在用冰冷的PBS洗涤一次后,用皿中的提取缓冲液裂解细胞。代表性数据如图1所示。

代表数据



图1.在小鼠成纤维细胞中诱导TNF-反应性基因的时间过程。 和 Cxcl 使用实时RT-PCR在加入10ng/ml的TNF之后的不同时间点检查来自野生型(WT)和三四分子(TTP) - 缺陷(KO)小鼠的血清剥夺的MEF中的mRNA。显示的结果是五个独立实验的平均值±SEM,并且数据表示为相对于时间0时的WT的倍数变化;在归一化为内部对照,Actb mRNA后,将该值设置为1。表示通过配对Student t检验确定的WT和KO平均值之间的统计学差异(* P <0.05,WT对KO)。经过许可修改自Qiu em et al。(2015)。

食谱

  1. 冲洗介质
    DMEM
    1%青霉素 - 链霉素
  2. 完成媒介
    DMEM
    10%FBS
    1%青霉素 - 链霉素 2mM谷氨酰胺
  3. 血清饥饿培养基
    DMEM
    0.5%FBS
    1%青霉素 - 链霉素 2mM谷氨酰胺

致谢

该方案改编自先前发表的研究,Lai等人(2006)和Horner等人(2009),并且用于Qiu等人。 (2015)。我们感谢Dee Wenzel为畜牧业提供支持,Christopher McGee为活体动物成像提供帮助,Drs。 Melissa Wells和Diana Cruz-Topete对协议的意见。这项研究由国立环境卫生科学研究所,国立卫生研究院的校内研究计划支持。

参考文献

  1. Horner,TJ,Lai,WS,Stumpo,DJ和Blackshear,PJ(2009)。 
  2. Lai,WS,Parker,JS,Grissom,SF,Stumpo,DJ和Blackshear,PJ(2006)。  通过在TTP缺陷成纤维细胞中稳定转录物的全面分析鉴定的三四脯氨酸(TTP)的新型mRNA靶标。 :9196-9208。
  3. Qiu,LQ,Lai,WS,Bradbury,A.,Zeldin,DC和Blackshear,PJ(2015)。  三四脯氨酸(TTP)协调调节原发性和继发性细胞对促炎症刺激的反应。 Leukoc Biol 97(4):723-736。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Qiu, L. Q., Lai, W. S., Stumpo, D. J. and Blackshear, P. J. (2016). Mouse Embryonic Fibroblast Cell Culture and Stimulation. Bio-protocol 6(13): e1859. DOI: 10.21769/BioProtoc.1859.
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