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Exit from Pluripotency Assay of Mouse Embryonic Stem Cells
小鼠胚胎干细胞退出多能性测定实验   

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参见作者原研究论文

本实验方案简略版
The Journal of Cell Biology
Feb 2017

Abstract

A novel method to assess the dissolution of the core pluripotency transcription-factor circuit of mouse Embryonic Stem Cells (mESCs) has been developed (Ying et al., 2003; Betschinger et al., 2013). In order to efficiently identify genes essential for the break-down of the pluripotency network in mutant mESCs with proliferation defects, we adapted this ‘exit from pluripotency assay’ (Bodak et al., 2017; Cirera-Salinas et al., 2017). The protocol described here has been successfully applied to several mESC lines and is easily transposable from one laboratory to another.

Keywords: Mouse embryonic stem cells (小鼠胚胎干细胞), Pluripotency (多能性), Commitment (细胞定向), 2i media (2i培养基), RNA interference (RNA干扰), Alkaline phosphatase (碱性磷酸酶), in vitro assay (体外实验)

Background

For decades, scientists have tried to identify the mechanisms underlying the differentiation potential of mESCs with general (e.g., Embryoïd Body) or directed (e.g., Neuronal Precursor Cells) differentiation protocols. Recently, the 2i culture media was discovered allowing the captivation of the naïve stem cells state in vitro (Ying et al., 2008). Betschinger and colleagues took advantage of the medium and developed a new ‘exit from pluripotency’ assay allowing the identification of novel factors involved in the commitment of mESCs (Betschinger et al., 2013). Briefly, mESCs maintained in 2i media conditions, are placed for two days in permissive media. Subsequently, the 2i media is reintroduced allowing only the survival of naïve mESCs. In this assay, wild type (WT) mESCs commit to differentiation during the two days of permissive media and die after reintroduction of 2i medium. Indeed, indicating that only two days of permissive media are sufficient to break-down the pluripotency network and commit to differentiation. Unfortunately, mutant mESCs for RNA interference pathways, i.e., Dicer and Dgcr8 genes, proliferate much slower than their WT counterparts making the assessment of the exit from pluripotency in two days with the original protocol less suitable. We decided to extend the presence of the cells in the permissive media to four days and then to reintroduce the 2i media for three more days. Only cells that do not commit during the four days in permissive media conditions will be able to survive and proliferate during the final three days in 2i media. Finally, cell survival and stemness are measured with Alkaline Phosphatase (AP) staining, as in the initial protocol.

Materials and Reagents

  1. 10 cm plate (TPP Techno Plastic Products, catalog number: 93100 )
  2. 6-well plate (TPP Techno Plastic Products, catalog number: 92006 )
  3. 10 ml pipettes (Bioswisstec, catalog number: 515210 )
  4. 5 ml pipettes (Bioswisstec, catalog number: 515205 )
  5. 15 ml Falcon tube (Greiner Bio One International, catalog number: 188271 )
  6. Glass Pasteur pipette (HUBERLAB, catalog number: 1.1127.01 )
  7. Mouse embryonic stem cells (E14TG2a mESC line obtained from ATCC: ES-E14TG2a) (ATCC, catalog number: CRL-1821 )
  8. Phosphate-buffered saline (PBS) 1x (Thermo Fisher Scientific, GibcoTM, catalog number: 10010015 )
  9. Trypsin-EDTA 0.05% (Thermo Fisher Scientific, GibcoTM, catalog number: 25300054 )
  10. Alkaline phosphatase Kit (Sigma-Aldrich, catalog number: 86R-1KT )
  11. ddH2O (Sigma-Aldrich, catalog number: 99053 )
    Note: This product has been discontinued.
  12. Gelatin (Sigma-Aldrich, catalog number: G1890 )
  13. Dulbecco’s modified Eagle medium (DMEM) high glucose (Sigma-Aldrich, catalog number: D6429 )
  14. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10270106 )
  15. Leukemia inhibitory factor (LIF) 10 millions unit/ml (Merck, catalog number: ESG1107 )
  16. 2-Mercaptoethanol (βM) 50 mM (Thermo Fisher Scientific, GibcoTM, catalog number: 31350010 )
  17. Mixture of penicillin and streptomycin (PS) (Sigma-Aldrich, catalog number: P0781 )
  18. N2B27 (Takara Bio, catalog number: Y40002 )
  19. MAPK/ERK inhibitor 0.4 μM (PD03) (Stemolecule PD0325901) (STEMCELL Technologies, catalog number: 72184 )
  20. GSK3β inhibitor 3 μM (Chiron) (Stemolecule CHIR99021) (STEMCELL Technologies, catalog number: 72054 )
  21. Acetone (Merck, catalog number: 1.00014.1000 )
  22. Formaldehyde 37% (Sigma-Aldrich, catalog number: 47608-1L-F )
  23. 0.2% gelatin solution (see Recipes)
  24. MES medium (see Recipes)
  25. MEF medium (see Recipes)
  26. 2i + LIF medium (see Recipes)
  27. Fixative solution (see Recipes)
  28. Substrate solution (see Recipes)

Equipment

  1. HeracallTM 150i incubator (37 °C and 8% CO2) (Thermo Fischer Scientific, Thermo ScientificTM, model: HeracallTM 150i )
  2. Centrifuge (Eppendorf, model: 5810 )
  3. Tissue culture hood (FASTER, model: Safe FAST Premium 209, catalog number: F00024900000 )
  4. Millipore ScepterTM 2.0 cell counter (EMD Millipore, model: ScepterTM 2.0, catalog number: PHCC00000 ) (or any cell counter)
  5. Inverted microscope (Nikon Instruments, model: Eclipse TS100 )

Software

  1. ImageJ software 1.48v
  2. Adobe Photoshop CS6
  3. GraphPad Prism 6.0a software

Procedure

  1. Maintenance of mESCs
    1. mESCs are routinely cultured on 10 cm dish pre-coated with 0.2% gelatin (see Recipes) in 10 ml of MES medium (see Recipes), in an 8% CO2 incubator at 37 °C (Figure1A).
    2. Medium is changed every other day.
      Note: Special attention should be taken to avoid confluency (> 70%) of mESCs (Figure 1B) in maintenance cultures as it might result in detrimental differentiation of the cells.


      Figure 1. Typical morphologies of mESCs. Feeder-free undifferentiated mESCs are shown at low 20-30% (A) and high 60-70% (B) density. Healthy, emergent mESCs grow as tightly formed, refractile colonies throughout passages (A). Over confluency (> 70%) of mESCs (B) creates flat colony morphology and potential differentiation.

  2. Exit from pluripotency assay
    1. Coat a 6-well plate with 0.2% gelatin (500 μl per well) and verify that the gelatin is well distributed. Place the plate in the incubator.
    2. Remove the media from a 50% confluent mESCs 10 cm dish. Wash once with 4 ml of 1x PBS. Add 2 ml of trypsin-EDTA to the cells and verify that the trypsin is well distributed. Place the cells back in the incubator for 5 min. Verify under the microscope that the mESC colonies are detached from the dish.
    3. Add 4 ml of MEF media (see Recipes) to the dish to inactivate the trypsin. Use a 5 ml pipette gently up and down to achieve single cells and move them in a 15 ml Falcon tube.
      Note: Try to achieve a single cell suspension and be careful to avoid too many bubbles.
    4. Count the cell density using a cell counter or equivalent method.
    5. Centrifuge 2 x 104 cells in a new tube at 190 x g for 5 min.
      Note: All centrifugations are carried at room temperature.
    6. Carefully resuspend the cell pellet using 2 ml of 2i + LIF media (see Recipes).
    7. Plate the 2 x 104 mESCs in 2 ml of 2i + LIF medium in the 6-well pre-coated plate with 0.2% gelatin. Medium is changed every other day.
      Notes:
      1. Do not forget to remove the gelatin before plating the cells.
      2. The day on which the culture is started is designated as Day 0.
    8. After 3 days, aspirate the 2i + LIF media without perturbing the mESC colonies (Day 3) and add N2B27 media (without LIF nor inhibitors) for 4 days (permissive media). Medium is changed every other day.
    9. At Day 7, remove the N2B27 (permissive media) media without perturbing the mESC colonies and reintroduce 2i + LIF media for 3 additional days. Medium is changed every other day.
    10. On Day 10 alkaline phosphatase staining can be performed (Figure 2).


      Figure 2. Alkaline phosphatase (AP) staining after exit from pluripotency assay. A. Whole 6-well after AP staining of wildtype mESCs; B. Wildtype mESCs are colorless and/or differentiated; C. Whole 6-well after AP staining of Dgcr8_KO mESCs; D. Dgcr8_KO mESCs cannot exit pluripotency and appear purple and undifferentiated; E. Number of positive clonal AP staining of cells after exit from pluripotency assay.

  3. Alkaline phosphatase staining
    1. Aspirate the 2i medium with a Pasteur pipette.
    2. Wash once the cells with 1 ml of 1x PBS per well.
      Note: Be careful not to wash off the colonies from the plate.
    3. Fix the cells carefully for 45 sec with 0.5 ml fixative solution (see Recipes).
      Note: Prolonged fixation can destroy alkaline phosphatase activity.
    4. Wash the cells twice with 0.5 ml ddH2O.
      Note: Be careful not to wash out the colonies from the plate.
    5. Add 0.5 ml substrate solution (see Recipes) for 15 min at room temperature in the dark (either drawer or aluminum foil).
    6. Wash with 0.5 ml ddH2O.
    7. Let the plate air dry overnight in a drawer.

  4. Clonal alkaline phosphatase positive number quantification
    1. Entire 6-well plates used for alkaline phosphatase staining are scanned with a printer/scanner machine to capture the total plate area in a single image (Figures 2A and 2C). More detailed images are also acquired with the microscope to better observe the morphology and alkaline phosphatase staining intensity in the different cell lines (Figures 2B and 2D).
    2. Scanned PDF images are converted into TIFF images with Adobe Photoshop CS6.
    3. Images are processed using ImageJ software 1.48v as follows:
      1. Number of alkaline phosphatase positive colonies is calculated on threshold intensity of inverted regions that are user–selected (full well–identical areas between conditions)
      2. Using the Analyze Particles tool (default parameters, ImageJ) the total number of alkaline phosphatase colonies is counted.
      3. Total number of alkaline phosphatase positive colonies is depicted in a graph as a clonal assay (Figure 2E).

Data analysis

Number of clonal AP positive colonies is expressed as ± SEM. Statistical differences can be measured by Student’s t-test. A value of P < 0.05 is considered significant. Data analysis can be performed using GraphPad Prism 6.0a software. AP staining must be performed in triplicate. The original research papers containing the aforementioned protocol are Cirera-Salinas et al., 2017 and Bodak et al., 2017.

Notes

The aforementioned protocol has been designed for RNAi mutant mESCs with strong proliferative defects. If other mouse ESC lines are used, we recommend to first adapting the cell lines to the 2i media. The adaption time varies from 3 days for good adherent cell lines, to 5-7 days if the cells have difficulties to attach to the plate. If the mESC line did not attach well, treat the plate with 0.2% gelatin for 24 h in the incubator before to plate them. Morphology of the cells is a good indicative for the adaptation as the cells become very round when adapted to the 2i media. Usually mESCs need 2 days in permissive media to exit from pluripotency. Finally, independently of the cell line used, 3 days in 2i media are necessary before staining with alkaline phosphatase.

Recipes

  1. 0.2% gelatin
    Dilute 1 g of gelatin from porcine skin with 500 ml of ddH2O, mix well and then autoclave solution


  2. MES medium


  3. MEF medium


  4. 2i + LIF medium


  5. Fixative solution (15 ml)
    12.25 ml acetone
    3.92 ml citrate (provided 25 mM solution from the Sigma-Aldrich kit)
    1.5 ml 37% formaldehyde
  6. Substrate solution (3 ml)
    2.81 ml ddH2O
    62.5 µl nitrite (provided by the Sigma-Aldrich kit)
    62.5 µl FRV (provided by the Sigma-Aldrich kit)
    62.5 µl of Naphthol (provided by the Sigma-Aldrich kit)
    Note: The order of the substrates nitrite (1st), FRV (2nd) and Naphthol (3rd) is important. Liquid has to be yellow before adding it to the cells.

Acknowledgments

This protocol was originally published as part of Cirera-Salinas et al. (2017). The authors wish to thank M. Bodak for fruitful discussions. D. C-S is supported by a Post-doctoral fellowship from the Peter and Traudl Foundation.

References

  1. Betschinger, J., Nichols, J., Dietmann, S., Corrin, P. D., Paddison, P. J. and Smith, A. (2013). Exit from pluripotency is gated by intracellular redistribution of the bHLH transcription factor Tfe3. Cell 153(2): 335-347.
  2. Bodak, M., Cirera-Salinas, D., Yu, J., Ngondo, R. P. and Ciaudo, C. (2017). Dicer, a new regulator of pluripotency exit and LINE-1 elements in mouse embryonic stem cells. FEBS Open Bio 7(2): 204-220.
  3. Cirera-Salinas, D., Yu, J., Bodak, M., Ngondo, R. P., Herbert, K. M. and Ciaudo C. (2017). Non-canonical function of DGCR8 controls mESCs exit from pluripotency. J Cell Biol.
  4. Ying, Q. L., Stavridis, M., Griffiths, D., Li, M. and Smith, A. (2003). Conversion of embryonic stem cells into neuroectodermal precursors in adherent monoculture. Nat Biotechnol 21(2): 183-186.
  5. Ying, Q. L., Wray, W., Nichols, J., Battle-Morera, L., Doble, B., Woodgett, J., Cohen, P. and Smith, A. (2008). The ground state of embryonic stem cell self-renewal. Nature 453(7194): 519-23.

简介

已经开发了评估小鼠胚胎干细胞(mESCs)的核心多能转录因子电路溶解的新方法(Ying等,2003; Betschinger等,2013)。 为了有效识别具有增殖缺陷的突变体mESCs中多能网络分解所必需的基因,我们调整了这种“多能性测定法”(Bodak等,2017; Cirera-Salinas等,2017)。 这里描述的方案已经成功应用于几个mESC系列,并且可以容易地从一个实验室转座到另一个实验室。
【背景】几十年来,科学家已经尝试确定基因与一般(例如胚胎体)或定向(例如,神经元前体细胞)分化方案的mESCs的分化潜能的机制。最近,发现2i培养基允许在体外俘获天真的干细胞(Ying et al。,2008)。 Betschinger及其同事利用了该媒介,并开发出了一种新的“多能性退出”分析方法,可以确定涉及mESCs承诺的新因素(Betschinger等,2013)。简言之,在2i媒体条件下维持的mESCs在许可媒介中放置两天。随后,2i媒体被重新引入,仅允许幼稚的mESCs的存活。在该测定中,野生型(WT)mESCs在允许的培养基的两天内进行分化,并在再次引入2i培养基后死亡。事实上,这表明只有两天的宽容媒体足以打破多能网络并致力于分化。不幸的是,用于RNA干扰途径的突变体mESC,即Dicer和Dgcr8基因,比其WT对应物增殖得慢得多,使得在两天内对多能性的退出进行评估,原始方案不合适。我们决定把许可媒介中的细胞存在四天,然后重新引入2i培养基三天。只有在许可媒体条件下的四天内不承诺的细胞才能够在最后三天在2i媒体中生存和增殖。最后,如初始方案,用碱性磷酸酶(AP)染色测量细胞存活和干细胞。

关键字:小鼠胚胎干细胞, 多能性, 细胞定向, 2i培养基, RNA干扰, 碱性磷酸酶, 体外实验

材料和试剂

  1. 10厘米板(TPP Techno Plastic Products,目录号:93100)
  2. 6孔板(TPP Techno Plastic Products,目录号:92006)
  3. 10ml移液器(Bioswisstec,目录号:515210)
  4. 5ml移液器(Bioswisstec,目录号:515205)
  5. 15ml Falcon管(Greiner Bio One International,目录号:188271)
  6. 玻璃巴斯德移液器(HUBERLAB,目录号:1.1127.01)
  7. 小鼠胚胎干细胞(得自ATCC:ES-E14TG2a的E14TG2a mESC系)(ATCC,目录号:CRL-1821)
  8. 磷酸盐缓冲盐水(PBS)1X(Thermo Fisher Scientific,Gibco TM,目录号:10010015)
  9. 胰蛋白酶-EDTA 0.05%(Thermo Fisher Scientific,Gibco TM,目录号:25300054)
  10. 碱性磷酸酶试剂盒(Sigma-Aldrich,目录号:86R-1KT)
  11. ddH 2 O(Sigma-Aldrich,目录号:99053)
    注意:本产品已停产。
  12. 明胶(Sigma-Aldrich,目录号:G1890)
  13. Dulbecco的改良Eagle培养基(DMEM)高葡萄糖(Sigma-Aldrich,目录号:D6429)
  14. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM,目录号:10270106)
  15. 白血病抑制因子(LIF)10百万单位/ ml(默克,目录号:ESG1107)
  16. 2-巯基乙醇(βM)50mM(Thermo Fisher Scientific,Gibco TM,目录号:31350010)
  17. 青霉素和链霉素(PS)的混合物(Sigma-Aldrich,目录号:P0781)
  18. N2B27(Takara Bio,目录号:Y40002)
  19. MAPK / ERK抑制剂0.4μM(PD03)(Stemolecule PD0325901)(STEMCELL Technologies,目录号:72184)
  20. GSK3β抑制剂3μM(Chiron)(Stemolecule CHIR99021)(STEMCELL Technologies,目录号:72054)
  21. 丙酮(Merck,目录号:1.00014.1000)
  22. 甲醛37%(Sigma-Aldrich,目录号:47608-1L-F)
  23. 0.2%明胶溶液(见配方)
  24. MES培养基(见食谱)
  25. MEF培养基(见食谱)
  26. 2i + LIF培养基(参见食谱)
  27. 固定溶液(参见食谱)
  28. 底物溶液(参见食谱)

设备

  1. Heracall TM 150i培养箱(37℃和8%CO 2)(Thermo Fischer Scientific,Thermo Scientific TM,型号:Heracall TM 150i)
  2. 离心机(Eppendorf,型号:5810)
  3. 组织文化罩(FASTER,型号:Safe FAST Premium 209,目录号:F00024900000)
  4. Millipore权杖 2.0细胞计数器(EMD Millipore,型号:Scepter TM 2.0,目录号:PHCC00000)(或任何细胞计数器)
  5. 倒置显微镜(Nikon Instruments,型号:Eclipse TS100)

软件

  1. ImageJ软件1.48v
  2. Adobe Photoshop CS6
  3. GraphPad Prism 6.0a软件

程序

  1. 维持mESCs
    1. 在37℃下在8%CO 2培养箱中将mESC常规培养在用0.2%明胶(参见食谱)预先包被在10ml MES培养基(参见食谱)中的10cm皿上(图1A )。
    2. 介质每隔一天更换一次。
      注意:应特别注意避免在维持培养中汇合(> 70%)的mESCs(图1B),否则可能导致细胞的有害分化。


      图1. mESCs的典型形态。无饲料未分化的mESC显示为低20-30%(A)和高60-70%(B)密度。健康的,紧急的mESCs生长形式紧密,遍及通过(A)的折射菌落。过度融合(> 70%)的mESCs(B)产生平坦的集落形态和潜在的分化。

  2. 退出多能测定
    1. 用0.2%明胶(500μl/孔)包被6孔板,并验证明胶分布良好。将板放在培养箱中。
    2. 从50%汇合的mESCs 10厘米盘中取出培养基。用4ml 1x PBS洗涤一次。向细胞中加入2ml胰蛋白酶-EDTA,验证胰蛋白酶分布良好。将细胞放回培养箱中5分钟。在显微镜下验证mESC菌落与盘分离。
    3. 加入4 ml的MEF培养基(参见食谱)到培养皿中以灭活胰蛋白酶。使用5ml移液器轻轻地上下实现单个细胞,并将它们移动到一个15毫升的Falcon管中。
      注意:尝试实现单细胞悬浮液,并小心避免太多气泡。
    4. 使用细胞计数器或等效方法计数细胞密度。
    5. 在新管中以200g离心2×10 4个细胞5分钟。
      注意:所有的离心机都是在室温下进行。
    6. 使用2ml 2i + LIF培养基小心地重悬细胞沉淀(参见食谱)。
    7. 在含有0.2%明胶的6孔预包被的平板中,将2×10 4细胞培养板放入2ml的2i + LIF培养基中。媒体每隔一天更换一次。
      注意:

      1. 不要忘记在电镀前清除明胶
      2. 文化开始的日子被指定为第0天。
    8. 3天后,吸入2i + LIF培养基而不扰乱mESC菌落(第3天),并加入N2B27培养基(不含LIF或抑制剂)4天(允许培养基)。媒体每隔一天更换一次。
    9. 在第7天,除去N2B27(许可培养基)培养基,而不干扰mESC菌落,并再次引入2i + LIF培养基3天。介质每隔一天更换一次。
    10. 在第10天,可以进行碱性磷酸酶染色(图2)

      图2.从多能性测定出来后的碱性磷酸酶(AP)染色。 :一种。野生型mESCs AP染色后6孔全部;野生型mESCs是无色和/或分化的; C.Dgcr8_KO mESCs AP染色后全6孔; D.Dgcr8_KO mESCs不能退出多能性,呈现紫色和未分化; E.从多能性测定出来后细胞的阳性克隆AP染色数。

  3. 碱性磷酸酶染色
    1. 用巴斯德吸管吸出2i培养基。
    2. 每孔用1ml 1×PBS清洗细胞。
      注意:注意不要从盘子上清除菌落。
    3. 用0.5ml固定溶液小心固定细胞45秒(参见食谱)。
      注意:长时间固定会破坏碱性磷酸酶活性。
    4. 用0.5ml ddH 2 O洗涤细胞两次。
      注意:注意不要从盘子里清除菌落。
    5. 在黑暗中(抽屉或铝箔)在室温下加入0.5ml底物溶液(参见食谱)15分钟。
    6. 用0.5ml ddH 2 O洗涤。
    7. 让盘子在抽屉里空气干燥。

  4. 克隆碱性磷酸酶阳性数量化
    1. 用打印机/扫描仪扫描用于碱性磷酸酶染色的整个6孔板以捕获单个图像中的总板面积(图2A和2C)。还可以用显微镜获取更详细的图像,以更好地观察不同细胞系中的形态和碱性磷酸酶染色强度(图2B和2D)。
    2. 扫描的PDF图像使用Adobe Photoshop CS6转换为TIFF图像。
    3. 使用ImageJ软件1.48v处理图像如下:
      1. 碱性磷酸酶阳性菌落的数量是用户选择的倒置区域的阈值强度(条件之间完全相同的区域)计算的
      2. 使用“分析粒子”工具(默认参数ImageJ)计算碱性磷酸酶菌落总数。
      3. 碱性磷酸酶阳性菌落的总数在图中作为克隆测定法进行描绘(图2E)

数据分析

克隆AP阳性菌落的数目表示为±SEM。统计学差异可以通过Student's 测试来测量。值 0.05被认为是显着的。可以使用GraphPad Prism 6.0a软件进行数据分析。 AP染色必须一式三份进行。包含上述协议的原始研究论文是Cirera-Salinas等人,2017和Bodak等人,2017。

笔记

上述方案已被设计用于具有强增殖缺陷的RNAi突变体mESC。如果使用其他鼠标ESC线,我们建议首先使细胞系适应2i介质。适应时间对于良好贴壁细胞系而言为3天,如果细胞难以附着在板上,则为5-7天。如果mESC系不适合,在培养箱中用0.2%明胶处理板24小时,然后将其平板。当细胞适应于2i培养基时,细胞的形态学是适应性的良好指示,因为细胞变得非常圆。通常,mESCs在许可培养基中需要2天才能从多能性中退出。最后,独立于使用的细胞系,在用碱性磷酸酶染色之前,需要3天的2i培养基。

食谱

  1. 0.2%明胶
    用500ml ddH 2 O稀释1g来自猪皮肤的明胶,充分混合,然后高压釜溶液


  2. MES中等


  3. MEF中等

  4. 2i + LIF媒体


  5. 固定溶液(15 ml)
    12.25毫升丙酮 3.92ml柠檬酸盐(提供来自Sigma-Aldrich试剂盒的25mM溶液)
    1.5 ml 37%甲醛
  6. 底物溶液(3ml)
    2.81ml ddH 2 O
    62.5μl亚硝酸盐(由Sigma-Aldrich试剂盒提供)
    62.5μlFRV(由Sigma-Aldrich试剂盒提供)
    62.5μlNapthol(由Sigma-Aldrich试剂盒提供)
    注意:底物亚硝酸盐(1st),FRV(2nd)和Napthol(3 rd )的顺序是重要的。在将细胞添加到细胞之前,液体必须是黄色的。

致谢

该协议最初是作为Cirera-Salinas等人的一部分出版的。 (2017)。作者希望感谢Bodak博士的富有成效的讨论。 D. C-S由Peter和Traudl基金会的博士后研究金支持。

参考

  1. Betschinger,J.,Nichols,J.,Dietmann,S.,Corrin,PD,Paddison,PJ和Smith,A.(2013)。&lt; a class =“ke-insertfile”href =“http: .ncbi.nlm.nih.gov / pubmed / 23582324“target =”_ blank“>从多能性退出是通过bHLH转录因子Tfe3的细胞内再分配来选择的。细胞 153(2) :335-347。
  2. Bodak,M.,Cirera-Salinas,D.,Yu,J.,Ngondo,RP and Ciaudo,C.(2017)。 Dicer,小鼠胚胎干细胞中多能性出口和LINE-1元素的新调节因子。 FEBS Open Bio 7(2):204-220。
  3. Cirera-Salinas,D.,Yu,J.,Bodak,M.,Ngondo,RP,Herbert,KM and Ciaudo C.(2017)。 DGCR8的非规范功能可以控制mESCs从多能性中退出。
  4. Ying,QL,Stavridis,M.,Griffiths,D.,Li,M. and Smith,A.(2003)。 将胚胎干细胞转化为粘附单一培养物中的神经外胚层前体。生物技术<21>(2):183-186。 br />
  5. Ying,QL,Wray,W.,Nichols,J.,Battle-Morera,L.,Doble,B.,Woodgett,J.,Cohen,P.和Smith,A.(2008)。胚胎干细胞自我更新的基础状态 自然 453(7194):519-23。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Cirera-Salinas, D. and Ciaudo, C. (2017). Exit from Pluripotency Assay of Mouse Embryonic Stem Cells. Bio-protocol 7(16): e2507. DOI: 10.21769/BioProtoc.2507.
  2. Cirera-Salinas, D., Yu, J., Bodak, M., Ngondo, R. P., Herbert, K. M. and Ciaudo C. (2017). Non-canonical function of DGCR8 controls mESCs exit from pluripotency. J Cell Biol.
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