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TGFβ Stimulation Assay
TGFβ刺激试验   

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Abstract

TGFβ is part of a growth factor superfamily which modulates cell growth, differentiation, adhesion, migration, ECM synthesis and apoptosis (Massague, 1998; Siegel and Massague, 2003). Free TGFβ binds to its high affinity TGFβ receptor, a receptor serine/threonine kinase, inducing phosphorylation of Smad2/3 which subsequently forms a complex with Smad4 to translocate to the nucleus where it interacts with multiple co-activators and repressors generating distinct transcriptional responses.
Indeed, TGFβ signaling shows a remarkable cellular context dependency and apparent multifunctionality: e.g. TGFβ is able to inhibit cell proliferation in many epithelial cells but can also enhance proliferation in fibroblasts and cell growth in endothelial cells (Guasch et al., 2007; Xiao et al., 2012); it enhances stem cell pluripotency, but promotes differentiation in other cells (Park, 2011); in cancer development it suppresses pre-malignant cell proliferation, but at the same time promotes conversion to a metastatic phenotype (Chaudhury and Howe, 2009).
The TGFβ stimulation assay monitors the responsiveness of cells to TGFβ. Upon TGFβ stimulation short-term effects such as Smad2 phosphorylation and long-term effects such as cell proliferation can be analyzed. The assay will be described for murine keratinocytes, where TGFβ strongly inhibits cell proliferation, but both assays are applicable for other cell types as well.

Materials and Reagents

  1. Cell line(s) of interest [here primary murine keratinocytes isolated as described in Montanez et al. (2007)]
  2. Antibodies
    1. pSmad2 (Merck KGaA, catalog number: AB3849 )
    2. Total Smad2/3 (Santa Cruz, catalog number: sc11769 )
    3. Goat anti–rabbit conjugated with horseradish peroxidase (HRP) (Bio-Rad Laboratories, catalog number: 172-1011 )
  3. Bovine serum albumin (BSA) fraction V (Carl Roth, catalog number: 8076 )
  4. Calcium chloride (CaCl2) (Carl Roth, catalog number: A119.1 )
  5. Chelex 100 resin (Bio-Rad Laboratories, catalog number: 143-2832 )
  6. Collagen I (PureCol, Advanced BioMatrix, catalog number: 5005-B )
  7. Fibronectin (Life Technologies, Gibco®, catalog number: 33016-015 )
  8. Protein standard (e.g. precision plus protein kaleidoscope standards) (Bio-Rad Laboratories, catalog number: 161-0375 )
  9. Fetal bovine serum (FBS) (Life Technologies, Gibco®, catalog number: 10270-106 )
  10. Minimum essential medium (MEM) (Sigma-Aldrich, catalog number: M8167 )
  11. Insulin (Sigma-Aldrich, catalog number: I5500 )
  12. Epidermal growth factor (EGF) (Sigma-Aldrich, catalog number: E4127 )
  13. Transferin (Sigma-Aldrich, catalog number: T8158 )
  14. Phosphoethanolamine (Sigma-Aldrich, catalog number: P0503 )
  15. Ethanolamine (Sigma-Aldrich, catalog number: E0135 )
  16. Hydrocortisone (Calbiochem, catalog number: 386698 )
  17. Trypsin powder (Life Technologies, Gibco®, catalog number: 27250-018 )
  18. L-Glutamine (Life Technologies, InvitrogenTM, catalog number: 25030-081 )
  19. Penicillin-streptomycin (pen-strep) (Life Technologies, InvitrogenTM, catalog number: 15070-063 )
  20. Click-iT® EdU Alexa Fluor® 488 Imaging Kit (Life Technologies, InvitrogenTM, catalog number: C10420 )
  21. Nonfat dried milk powder (AppliChem, catalog number: A0830 )
  22. PVDF membrane (e.g. Immobilon-P) (Merck KGaA, catalog number: IPVH00010 )
  23. Recombinant TGFβ1 (PeproTech, catalog number: 100-21 )
  24. Tween20 (Sigma-Aldrich, catalog number: P9416 )
  25. Sodium dodecyl sulfate (SDS) (Carl Roth, catalog number: 0183.1 )
  26. β-Mercaptoethanol (Carl Roth, catalog number: 4227.3 )
  27. Bromophenol blue (Carl Roth, cataog number: A512.1 )
  28. Sodium chloride (NaCl) (Carl Roth, catalog number: P029 )
  29. Sodium hydrogen carbonate (NHCO3) (Carl Roth, catalog number: 6885 )
  30. Sodium carbonate (Na2CO3) (Carl Roth, catalog number: A135 )
  31. Sodium deoxycholate (Sigma-Aldrich, catalog number: D6750 )
  32. di-Sodium hydrogen phosphate (Na2HPO4) (Carl Roth, catalog number: T876 )
  33. Glycine (Carl Roth, catalog number: 0079 )
  34. Glycerol (Carl Roth, catalog number: 4043.1 )
  35. Hydrochloric acid (HCl) (Sigma-Aldrich, catalog number: 258148 )
  36. Methanol (Sigma-Aldrich, catalog number: 322415 )
  37. Potassium chloride (KCl) (Carl Roth, catalog number: HN02.3 )
  38. Potassium dihydrogen phosphate (KH2PO4) (Carl Roth, catalog number: 3904 )
  39. Nonidet-P40 (NP-40) (e.g. IGEPAL CA-630) (Sigma-Aldrich, catalog number: I8896 )
  40. Protease inhibitor cocktail (e.g. cOmplete) (Roche, catalog number: 04693116001 )
  41. Phosphatase inhibitor cocktail 2 (Sigma-Aldrich, catalog number: P5726 )
  42. Phosphatase inhibitor cocktail 3 (Sigma-Aldrich, catalog number: P0044 )
  43. 2-Propanol (Carl Roth, catalog number: T902.1 )
  44. Tris-HCl (Carl Roth, catalog number: 9090.3 )
  45. Cell type specific growth (see Recipes)
  46. Cell type specific starving medium (see Recipes)
  47. Phosphate-buffered saline (PBS) (see Recipes)
  48. Cell lysis buffer supplemented with protease and phosphatase inhibitors (see Recipes)
  49. Standard SDS-PAGE running buffer (see Recipes)
  50. 2x Laemmli sample buffer (see Recipes)
  51. Western blot transfer buffer (see Recipes)
  52. TBS (see Recipes)
  53. TBS-T (see Recipes)
  54. Western blot stripping buffer (see Recipes)

Equipment

  1. 6-well plate and 10 cm dishes for cell culture
  2. Standard centrifuge to spin down cells and protein lysis
  3. Cell scraper (e.g. Corning cell scraper) (Sigma-Aldrich, catalog number: CLS3010 )
  4. Standard SDS-PAGE and Western blot equipment
  5. Cell culture Incubator 37 °C, 5% CO2 for standard cell culture conditions
  6. Flow cytometer (e.g. BD FACS Canto) using FlowJo as analysis software

Procedure

  1. Analysis of proliferation after TGFβ stimulation (long-term effect)
    Note: Each condition should be measured in technical triplicates. For keratinocytes one well of a 6 well plate is sufficient for FACS analysis but depending on the cell type, the assay can be performed with higher or lower amounts of cells. Proliferation is measured by uptake of the nucleotide analogue 5-ethynyl-2′-deoxyuridine (EdU) during cell cycle S-Phase and then detected by Click-iT chemistry. 5-brom-2’-deoxyuridine (BrdU) uptake can be used alternatively.
    1. Plate cell line of interest in complete medium in such a density, so that wells will reach a confluency of 70% the next day (1.2 x 105 cells/well for keratinocytes). For keratinocytes wells should be coated with coating medium for 30 min at 37 °C which should be removed before plating the cells).
    2. The next day medium is removed and (without any washing step) is replaced with cell culture medium (should be room temperature) supplemented with reduced serum concentration (here 5% FBS) and 0, 5 or 10 ng/ml TGFβ1 and continue culture under standard conditions (37 °C, 5% CO2).
    3. After 6 h add 10 µM EdU per well without changing the medium and continue to culture cells under standard conditions (37 °C, 5% CO2).
    4. Further incubate the cells for 8 h and then generate single cell suspension by trypsin digestion. For trypsin digestion remove the medium and wash cells once with PBS. Then add trypsin solution, incubate cells at standard condition (37 °C, 5% CO2) and as soon as cells have detached collect cells by adding complete medium. After spinning down the cells (5 min at 900 rpm, 78 x g) wash single cell suspension once with PBS before following Click-iT assay kit protocol.
      Note: The isolation time point can vary between cell types. It is recommended to determine in a pre-experiment a time point, where approximately 70% of the untreated cells of interest are EdU positive.
    5. Click-iT protocol (mainly follows manufacturers protocol which can be downloaded on the Invitrogen web site).
      1. Wash cells once with 1% BSA-PBS solution at room temperature.
        Note: Cell washes and buffer changes are performed by centrifugation of cell suspensions for 2 min at 2,100 rpm (380 x g), removal of the supernatant and gentle resuspension of the cells in the respective new buffer.
      2. Fix cells with 200 µl 3.7% formaldehyde in PBS (Component D in the kit) for 15 min at room temperature and permeabilize by buffer change to 200 µl 1x Click-iT saponin-based permeabilization solution (Component E in the kit) and incubate for 15 min.
      3. Prepare Click-iT staining solution according manufacturer protocol using PBS (see Click-iT reaction cocktail table) and incubate cells with this solution for 30 min in the dark at room temperature.
      4. Wash cells once with 500 µl Click-iT permeabilization and washing buffer, resuspend cells in 200 µl of the same buffer and immediately proceed to FACS measurement.
      5. To detect Alexa Fluor 488 for EdU labeling, use 488 nm excitation with a green emission filter 530/30 nm or similar (FL1 channel).
      6. Use unstained cells as negative control.
      7. Determine percentage of EdU positive cells, identifying all cells which have been in cell cycle S-phase during EdU incorporation (=proliferating cells).

  2. pSmad detection after TGFβ stimulation (short-term effect, or eminent effects)
    Note: Phosphorylation of Smad proteins is an immediate effect after TGFβ receptor activation of the canonical TGFβ signaling cascade and will occur in all cell types. Conversely the subsequent TGFβ downstream signaling can be highly diverse between different cell types.
    1. Plate for each condition one 10 cm dish. Cell of interest density should be chosen so that 10 cm dishes will reach a confluency of 70% the next day (for keratinocytes 2 x 106 cells/dish). For keratinocytes dishes should be coated with coating medium for 30 min at 37 °C which should be removed before plating the cells.
    2. The next day serum-starve the cells for several hours (2-4 h) in starving medium (e.g. Starving KGM) under standard cell culture conditions.
    3. Then add 5 ng/ml TGFβ1 to the starving medium and isolate cells after 0, 30, 60 and 120 min continued incubation at standard cell culture conditions.
    4. For cell isolation, wash cells once with PBS and lyse them in appropriate volume of lysis buffer (e.g. RIPA, for keratinocytes 120 µl per 10 cm dish) directly on the plate on ice. Collect cell lysates with a cell scraper and pipette up and down 5 times with a 200 µl tip for complete cell lysis. Then spin down cell lysates at full speed (10 min, 14,000 rpm, 16,800 x g) at 4 °C and isolate the supernatant (sample to run on SDS-page).
    5. Separate samples on a 10% SDS-PAGE using standard Western blot protocol considering that Smad2 migrates around 55-60 kDa. A lane with pre-stained protein standard (e.g. precision plus protein kaleidoscope standards) should be included (for keratinocytes, 20 µg of protein per lane was ideal.) After gel separation, samples were transferred onto a PVDF membrane, using over-night tank transfer and Dunn buffer. Recipes for 2x Laemmli sample buffer, standard SDS-PAGE running buffer, Dunn Carbonate buffer are included in the recipes section.
    6. For the detection of murine pSmad2 and total Smad2/3 the following antibodies and conditions were used:
      1. pSmad2:
        1. Block membrane for 1 h at room temperature with 5% milk TBS-T and incubate pSmad2 antibody 1:1,000 in 1% milk TBS-T over night at 4 °C.
        2. Wash membranes 3x 15 min with TBS-T and incubate with secondary antibody for 1 h at room temperature.
        3. Wash membranes 3x 15 min with TBS-T before detection.
      2. Total Smad2/3:
        1. Block membrane for 1 h at room temperature with 5% BSA TBS-T and incubate total Smad2/3 antibody 1:1,000 in 1% BSA TBS-T over night at 4 °C.
        2. Wash membranes 3x 15 min and incubate with secondary antibody for 1 h at room temperature.
        3. Wash membranes 3x 15 min before detection.
          Note: It is recommended to detect first pSmad2 and after membrane stripping to blot for total Smad2/3.
          For membrane stripping incubate membrane for 10 min in pre-heated (50 °C) stripping buffer at 50 °C. Then wash the membrane with ddH2O (4x 5 min) before reactivating the membrane with 2-propanol. Repeat the washing of the membranes (4x 5 min) before blocking.

Representative data



Figure 1. A. Representative FACS histogram of murine kerationcytes using Click-iT EdU Alexa Fluor 488 assay kit. Keratinocytes were incubated for 6 h with indicated TGFβ1 concentrations before addition of 10 µM EdU. Cells were analyzed after 8 h of EdU incorporation to detect proliferating cells. Note the decrease of proliferating cells with increasing TGFβ1 concentration. B. Representative Western bot for pSmad2 detection in murine kerationcytes after stimulation with 5 ng/ml TGFβ1 for indicated time points.

Recipes

  1. Cell type specific growth and starving medium as well as trypsin solution (shown for murine keratinocyte culture)
    1. Keratinocyte growth medium (KGM)
      Final working concentration
      Initial stock-concentration
      Vol.
      MEM

      500 ml
      5 µg/ml insulin
      5 mg/ml in 4mM HCl
      0.5 ml
      10 ng/ml EGF
      200 µg/ml in PBS
      25 µl
      10 µg/ml transferin
      5 mg/ml in PBS
      1 ml
      10 µM phosphoethanolamine
      10 mM in PBS
      0.5 ml
      10 µM ethanolamine
      10 mM in PBS
      0.5 ml
      0.36 µg/ml hydrocortisone
      5 mg/ml in ethanol
      36 µl
      1x glutamine
      100x
      5 ml
      1x pen-strep
      100x
      5 ml
      8% chelated FBS (Ca2+ free) (see Recipes)

      40 ml
      45 µM CaCl2 (sterile filtrated)
      100 mM
      225 µl
      Filter the mixture through 0.2 µm and stored at 4 °C for up to 1 month.
    2. Starving KGM
      Final working concentration
      Initial Stock-concentration
      Vol.
      MEM

      500 ml
      1x pen-strep
      100x
      5 ml
      45 µM CaCl2 (sterile filtrated)
      100 mM
      225 µl
      Filter the mixture through 0.2 µm and store at 4 °C for up to 1 month
    3. Keratinocyte trypsin (0.4%)
      Dissolve 0.4 g trypsin powder in 100 ml PBS and pass through a 0.2 µm filter for sterilization. It can be stored either at -20 °C for 1 year or at 4 °C for 1 month
      Avoid repeated freeze thaw cycles
    4. Chelated FBS
      Add hydrated Chelex 100 resin to FBS (20 g of resin for 40 ml of FBS) and stir for 1 h at 4 °C
      Further remove the resin by filtration and store chelated FBS at -20 °C
    5. Coating medium for keratinocytes
      Dilute Collagen I (30 µg/ml) and Fibronectin (10 µg/ml) in PBS directly before use
  2. Phosphate-buffered saline (PBS)
    Dissolve NaCl (137 mM) 8 g/L, KCl (2.7 mM) 0.2 g/L, Na2HPO4 (10 mM) 1.44 g/L, KH2PO4 (1.8 mM) 0.24 g/L in ddH2O and adjust pH to 7.4 with HCl
  3. Cell lysis buffer supplemented with protease and phosphatase inhibitors (for kerationcytes RIPA)
    For RIPA buffer dissolve Tris-HCl (50 mM, pH 7.4), NP-40 (1%), Na-deoxycholate (0.5%), SDS (0.1%), NaCl (150 mM) and EDTA (2 mM)
    (Prior to use add protease inhibitors and phosphatase inhibitor 2 and 3)
  4. Standard SDS-PAGE running buffer
    To prepare 10x running buffer dissolve Tis-HCl 30 g/L, Glycine 144 g/L and 10 g/L SDS in ddH2O
    Dilute to 1x before use
  5. 2x Laemmli sample buffer
    Dissolve Tris-HCl (100 mM, pH 6.8), 20% glycerol, 2% SDS and 0.01% bromophenol blue in ddH2O
    Add β-mercaptoethanol (4%) just before use
  6. Western blot transfer buffer (e.g. Dunn Carbonate buffer)
    Here Dunn Carbonate buffer is used
    For 1x Dunn Carbonate Buffer dissolve NaHCO3 (10mM) 0.84 g/L, Na2CO3 (3 mM) 0.318 g/L and methanol (20%) 200 ml in ddH2O
  7. TBS
    Dissolve Tris-HCl (50 mM) 6.05 g/L and NaCl (150 mM) 8.76 g/L in ddH2O and adjust pH to 7.5 with HCl
  8. TBS-T
    TBS supplemented with 0.1% Tween20
  9. Western blot stripping buffer
    2% SDS
    62.5 mM Tris-HCl (pH 6.7)
    100 mM β-Mercaptoethanol (add only directly before use)

Acknowledgments

This work was funded by the Max Planck Society.

References

  1. Chaudhury, A. and Howe, P. H. (2009). The tale of transforming growth factor-beta (TGFbeta) signaling: a soigne enigma. IUBMB Life 61(10): 929-939.
  2. Guasch, G., Schober, M., Pasolli, H. A., Conn, E. B., Polak, L. and Fuchs, E. (2007). Loss of TGFbeta signaling destabilizes homeostasis and promotes squamous cell carcinomas in stratified epithelia. Cancer Cell 12(4): 313-327.
  3. Massague, J. (1998). TGF-beta signal transduction. Annu Rev Biochem 67: 753-791.
  4. Montanez, E., Piwko-Czuchra, A., Bauer, M., Li, S., Yurchenco, P. and Fassler, R. (2007). Analysis of integrin functions in peri-implantation embryos, hematopoietic system, and skin. Methods Enzymol 426: 239-289.
  5. Park, K. S. (2011). Tgf-Beta family signaling in embryonic stem cells. Int J Stem Cells 4(1): 18-23.
  6. Siegel, P. M. and Massague, J. (2003). Cytostatic and apoptotic actions of TGF-beta in homeostasis and cancer. Nat Rev Cancer 3(11): 807-821.
  7. Xiao, L., Du, Y., Shen, Y., He, Y., Zhao, H. and Li, Z. (2012). TGF-beta 1 induced fibroblast proliferation is mediated by the FGF-2/ERK pathway. Front Biosci (Landmark Ed) 17: 2667-2674.

简介

TGFβ是调节细胞生长,分化,粘附,迁移,ECM合成和凋亡的生长因子超家族的一部分(Massague,1998; Siegel和Massague,2003)。游离TGFβ结合到其高亲和力TGFβ受体,一种受体丝氨酸/苏氨酸激酶,诱导Smad2/3的磷酸化,随后与Smad4形成复合物转移到细胞核,其中它与多种共激活剂和阻遏物相互作用产生不同的转录反应。实际上,TGFβ信号传导显示出显着的细胞环境依赖性和表观多功能性:例如TGFβ能够抑制许多上皮细胞中的细胞增殖,但也可以增强成纤维细胞中的增殖和内皮细胞中的细胞生长(Guasch等人,2007; Xiao等人,2012年);它增强干细胞多能性,但促进其他细胞的分化(Park,2011);在癌症发展中,它抑制恶变前细胞增殖,但同时促进转移到转移表型(Chaudhury和Howe,2009)。
TGFβ刺激测定监测细胞对TGFβ的反应性。在TGFβ刺激时,可以分析短期效应例如Smad2磷酸化和长期效应例如细胞增殖。将描述用于小鼠角质形成细胞的测定,其中TGFβ强烈抑制细胞增殖,但是这两种测定也适用于其它细胞类型。

材料和试剂

  1. 感兴趣的细胞系[如Montanez et al。(2007)所述分离的原代小鼠角质形成细胞]
  2. 抗体
    1. pSmad2(Merck KGaA,目录号:AB3849)
    2. Total Smad2/3(Santa Cruz,目录号:sc11769)
    3. 与辣根过氧化物酶(HRP)缀合的山羊抗兔(Bio-Rad Laboratories,目录号:172-1011)
  3. 牛血清白蛋白(BSA)级分V(Carl Roth,目录号:8076)
  4. 氯化钙(CaCl 2)(Carl Roth,目录号:A119.1)
  5. Chelex 100树脂(Bio-Rad Laboratories,目录号:143-2832)
  6. 胶原I(PureCol,Advanced BioMatrix,目录号:5005-B)
  7. 纤连蛋白(Life Technologies,Gibco ,目录号:33016-015)
  8. 蛋白质标准品(例如精密加蛋白万花筒标准品)(Bio-Rad Laboratories,目录号:161-0375)
  9. 胎牛血清(FBS)(Life Technologies,Gibco ,目录号:10270-106)
  10. 最低必需培养基(MEM)(Sigma-Aldrich,目录号:M8167)
  11. 胰岛素(Sigma-Aldrich,目录号:I5500)
  12. 表皮生长因子(EGF)(Sigma-Aldrich,目录号:E4127)
  13. 转移酶(Sigma-Aldrich,目录号:T8158)
  14. 磷酸乙醇胺(Sigma-Aldrich,目录号:P0503)
  15. 乙醇胺(Sigma-Aldrich,目录号:E0135)
  16. 氢化可的松(Calbiochem,目录号:386698)
  17. 胰蛋白酶粉(Life Technologies,Gibco ,目录号:27250-018)
  18. L-谷氨酰胺(Life Technologies,Invitrogen TM ,目录号:25030-081)
  19. 青霉素 - 链霉素(pen-strep)(Life Technologies,Invitrogen TM,目录号:15070-063)
  20. Click-iT EdU Alexa Fluor 488 Imaging Kit(Life Technologies,Invitrogen TM ,目录号:C10420)
  21. 脱脂奶粉(AppliChem,目录号:A0830)
  22. PVDF膜(例如Immobilon-P)(Merck KGaA,目录号:IPVH00010)
  23. 重组TGFβ1(PeproTech,目录号:100-21)
  24. Tween20(Sigma-Aldrich,目录号:P9416)
  25. 十二烷基硫酸钠(SDS)(Carl Roth,目录号:0183.1)
  26. β-Mercaptoethanol(Carl Roth,目录号:4227.3)
  27. 溴酚蓝(Carl Roth,catataog number:A512.1)
  28. 氯化钠(NaCl)(Carl Roth,目录号:P029)
  29. 碳酸氢钠(NHCO 3)(Carl Roth,目录号:6885)
  30. 碳酸钠(Na 2 CO 3)(Carl Roth,目录号:A135)
  31. 脱氧胆酸钠(Sigma-Aldrich,目录号:D6750)
  32. 磷酸氢二钠(Na 2 HPO 4)(Carl Roth,目录号:T876)
  33. 甘氨酸(Carl Roth,目录号:0079)
  34. 甘油(Carl Roth,目录号:4043.1)
  35. 盐酸(HCl)(Sigma-Aldrich,目录号:258148)
  36. 甲醇(Sigma-Aldrich,目录号:322415)
  37. 氯化钾(KCl)(Carl Roth,目录号:HN02.3)
  38. 磷酸二氢钾(KH 2 PO 4)(Carl Roth,目录号:3904)
  39. Nonidet-P40(NP-40)(例如IGEPAL CA-630)(Sigma-Aldrich,目录号:I8896)
  40. 蛋白酶抑制剂混合物(例如,cOmplete)(Roche,目录号:04693116001)
  41. 磷酸酶抑制剂混合物2(Sigma-Aldrich,目录号:P5726)
  42. 磷酸酶抑制剂混合物3(Sigma-Aldrich,目录号:P0044)
  43. 2-丙醇(Carl Roth,目录号:T902.1)
  44. Tris-HCl(Carl Roth,目录号:9090.3)
  45. 细胞类型特异性生长(参见配方)
  46. 细胞类型特异性饥饿培养基(参见配方)
  47. 磷酸盐缓冲盐水(PBS)(见配方)
  48. 补充有蛋白酶和磷酸酶抑制剂的细胞裂解缓冲液(参见Recipes)
  49. 标准SDS-PAGE运行缓冲液(参见配方)
  50. 2x Laemmli样品缓冲液(参见配方)
  51. Western印迹转移缓冲液(参见配方)
  52. TBS(参见配方)
  53. TBS-T(参见配方)
  54. Western印迹剥离缓冲液(参见配方)

设备

  1. 6孔板和10cm皿用于细胞培养
  2. 标准离心机离心细胞和蛋白质裂解
  3. 细胞刮刀(例如Corning细胞刮刀)(Sigma-Aldrich,目录号:CLS3010)
  4. 标准SDS-PAGE和Western印迹设备
  5. 细胞培养37℃,5%CO 2用于标准细胞培养条件
  6. 使用FlowJo作为分析软件的流式细胞仪(例如BD FACS Canto)

程序

  1. TGFβ刺激后的增殖分析(长期效应)
    注意:每个条件都应该用技术三次测量。 对于角质形成细胞,6孔板的一个孔足以用于FACS分析,但是取决于细胞类型,可以用更高或更低量的细胞进行测定。 通过在细胞周期S期期间摄取核苷酸类似物5-乙炔基-2'-脱氧尿苷(EdU)并随后通过Click-iT化学检测来测量增殖。 可以替代地使用5-溴-2'-脱氧尿苷(BrdU)摄取。
    1. 平板细胞系在完全培养基中的这种密度,所以 孔将达到第二天70%的汇合(对于角质形成细胞为1.2×10 5个细胞/孔)。 对于角质形成细胞,应该涂覆孔 与包被培养基在37℃下30分钟,应该之前删除 电镀细胞)。
    2. 第二天除去培养基(无 任何洗涤步骤)替换为细胞培养基(应为室 温度),补充有降低的血清浓度(此处为5%FBS)   和0,5或10ng/mlTGFβ1并继续在标准下培养 条件(37℃,5%CO 2)。
    3. 6小时后,每孔加入10μMEdU 而不改变培养基并继续培养细胞 标准条件(37℃,5%CO 2)。
    4. 进一步孵育细胞 8小时,然后通过胰蛋白酶消化产生单细胞悬浮液。 对于胰蛋白酶消化,去除培养基并用PBS洗涤细胞一次。 然后加入胰蛋白酶溶液,在标准条件(37℃, 5%CO 2),并且一旦细胞分离,通过加入收集细胞 完全培养基。 在使细胞离心(在900rpm下5分钟,78× g )用PBS洗涤单细胞悬液一次,然后使用Click-iT 测定试剂盒方案。
      注意:隔离时间点可以有所不同   细胞类型。 建议在预实验中确定一个时间 点,其中约70%的未处理的目的细胞是 EdU积极。
    5. Click-iT协议(主要遵循制造商协议,可在Invitrogen网站上下载)。
      1. 在室温下用1%BSA-PBS溶液洗涤细胞一次 注意:通过离心进行细胞洗涤和缓冲液更换   细胞悬浮液以2,100rpm(380×g)离心2分钟, 上清液和细胞在各自的新的缓慢重悬浮 缓冲区。
      2. 用200μl3.7%甲醛的PBS溶液(成分   D在试剂盒中)在室温下15分钟,并通过缓冲液透化   更换为200μl1x Click-iT皂苷基透化溶液 (试剂盒中的组分E)并孵育15分钟
      3. 准备 Click-iT染色溶液,根据制造商方案使用PBS (参见Click-iT反应混合物表)并用此孵育细胞 溶液在室温黑暗中30分钟
      4. 洗涤细胞 一次用500μlClick-iT透化和洗涤缓冲液,重悬   细胞在200μl的相同缓冲液中,并立即进行FACS 测量
      5. 要检测Alexa Fluor 488进行EdU标记,请使用488 nm激发用绿色发射滤光片530/30 nm或类似物(FL1 通道)。
      6. 使用未染色的细胞作为阴性对照。
      7. 确定EdU阳性细胞的百分比,识别所有细胞 已经在EdU掺入期间处于细胞周期S期(=增殖   细胞)。

  2. TGFβ刺激后的pSmad检测(短期效应或显着效应)
    注意:Smad蛋白的磷酸化是TGFβ受体激活经典TGFβ信号级联后的即时效应,并且将在所有细胞类型中发生。 相反,随后的TGFβ下游信号传导在不同细胞类型之间可以是高度不同的。
    1. 板为每个条件一个10厘米的菜。 感兴趣的细胞密度应该   选择10厘米的菜肴将达到70%的汇合 天(对于角质形成细胞2×10 6个细胞/皿)。 对于角质形成细胞培养皿 应在37℃下用包被培养基包被30分钟,这应该是   在电镀细胞前移除
    2. 第二天血清饥饿 细胞在饥饿培养基(例如饥饿KGM)中培养几小时(2-4小时) 在标准细胞培养条件下
    3. 然后加入5ng/mlTGFβ1   饥饿培养基和分离细胞在0,30,60和120分钟后 在标准细胞培养条件下继续温育。
    4. 对于 细胞分离,用PBS洗涤细胞一次并在适当的溶液中裂解 体积的裂解缓冲液(例如,RIPA),对于角质形成细胞,每10cm为120μl 盘)直接在冰上的板上。 用细胞收集细胞裂解物 刮刀和移液器向上和向下5次与200微升提示完成 细胞裂解。 然后以全速(10分钟,14,000rpm)离心细胞裂解物 rpm,16,800×g),并分离上清液(样品运行) SDS-page)。
    5. 使用标准在10%SDS-PAGE上分离样品 Western印迹方案考虑Smad2迁移约55-60 kDa。 一个   带有预染色的蛋白质标准品(例如,精密加蛋白质) 万花筒标准)(对于角质形成细胞,20μg 蛋白质每道是理想的。)凝胶分离后,样品 转移到PVDF膜上,使用过夜罐转移和 Dunn缓冲液。 2x Laemmli样品缓冲液,标准SDS-PAGE的配方 运行缓冲液,Dunn碳酸盐缓冲液包括在配方中 部分
    6. 对于鼠pSmad2和总Smad2/3的检测,使用以下抗体和条件:
      1. pSmad2:
        1. 在室温下用5%牛奶TBS-T封闭膜1小时 在4℃下在1%牛奶TBS-T中孵育pSmad2抗体1:1000过夜。
        2. 用TBS-T洗涤膜3×15分钟,并与第二抗体在室温下孵育1小时。
        3. 在检测前用TBS-T洗涤膜3×15分钟。
      2. 总Smad2/3:
        1. 在室温下用5%BSA TBS-T封闭膜1小时 在1%BSA TBS-T中将总Smad2/3抗体1:1,000孵育过夜 C。
        2. 洗涤膜3×15分钟,并与第二抗体在室温下孵育1小时。
        3. 检测前3x 15分钟洗膜。
          注意:建议检测第一个pSmad2,并且膜剥离后,检测总Smad2/3。
          对于膜剥离,在预热(50℃)下孵育膜10分钟 ℃)剥离缓冲液在50℃。 然后用ddH 2 O(4×5)洗涤膜 min),然后用2-丙醇再活化膜。 重复 洗涤膜(4×5分钟),然后封闭。

代表数据



图1。 A.使用Click-iT EdU Alexa Fluor 488测定试剂盒的小鼠血细胞的代表性FACS直方图。将角质形成细胞与指示的TGFβ1浓度孵育6小时,然后加入10μMEdU。在8小时的EdU掺入后分析细胞以检测增殖细胞。注意增殖细胞随TGFβ1浓度增加的减少。 B.在用5ng/mlTGFβ1刺激指定的时间点后,在鼠充血细胞中检测pSmad2的代表性Western bot。

食谱

  1. 细胞类型特异性生长和饥饿培养基以及胰蛋白酶溶液(显示用于鼠角质形成细胞培养物)
    1. 角质形成细胞生长培养基(KGM)
      最终工作浓度
      初始库存浓度
      卷。
      MEM

      500 ml
      5μg/ml胰岛素
      5mg/ml在4mM HCl中 0.5 ml
      10 ng/ml EGF
      200μg/ml,在PBS中
      25μl
      10μg/ml转移蛋白
      5mg/ml在PBS中 1 ml
      10μM磷酸乙醇胺 10mM在PBS中
      0.5 ml
      10μM乙醇胺
      10mM在PBS中
      0.5 ml
      0.36μg/ml氢化可的松
      5mg/ml的乙醇溶液 36微升
      1×谷氨酰胺
      100x
      5 ml
      1x pen-strep
      100x
      5 ml
      8%螯合的FBS(Ca 2+ +游离)(参见配方)
      40 ml
      45μMCaCl 2(无菌过滤)
      100 mM
      225微升
      将混合物过滤通过0.2μm,并在4℃下储存至多1个月
    2. 饥饿KGM
      最终工作浓度
      初始库存浓度
      卷。
      MEM

      500 ml
      1x pen-strep
      100x
      5 ml
      45μMCaCl 2(无菌过滤)
      100 mM
      225微升
      将混合物过滤通过0.2μm并在4℃下储存最多1个月
    3. 角质形成细胞胰蛋白酶(0.4%)
      将0.4g胰蛋白酶粉末溶解在100ml PBS中,并通过0.2μm 过滤器灭菌。 它可以在-20°C储存1年 或在4℃下1个月
      避免反复冻融周期
    4. Chelated FBS
      将水合Chelex 100树脂加入FBS(20g树脂,对于40ml FBS) 并在4℃下搅拌1小时 进一步通过过滤除去树脂 将螯合FBS储存在-20℃下
    5. 角质形成细胞的包被介质
      在使用前,立即稀释胶原I(30μg/ml)和纤连蛋白(10μg/ml)
  2. 磷酸盐缓冲盐水(PBS)
    将NaCl(137mM)8g/L,KCl(2.7mM)0.2g/L,Na 2 HPO 4(10mM)1.44g/L,KH 在ddH 2 O 2中的2×PO 4(1.8mM)0.24g/L,用HCl调节pH至7.4,
  3. 补充有蛋白酶和磷酸酶抑制剂(对于RIPA))的细胞裂解缓冲液 对于RIPA缓冲液,溶解Tris-HCl(50mM,pH 7.4),NP-40(1%),脱氧胆酸钠(0.5%),SDS(0.1%),NaCl(150mM) /> (使用前加蛋白酶抑制剂和磷酸酶抑制剂2和3)
  4. 标准SDS-PAGE运行缓冲液
    为了制备10x运行缓冲液,溶解ddH 2 O中的Tis-HCl 30g/L,甘氨酸144g/L和10g/L SDS。
    使用前稀释至1x/
  5. 2x Laemmli样品缓冲液
    在ddH 2 O中溶解Tris-HCl(100mM,pH 6.8),20%甘油,2%SDS和0.01%溴酚蓝。 在使用前加入β-巯基乙醇(4%)
  6. Western印迹转移缓冲液(例如Dunn碳酸盐缓冲液)
    这里使用Dunn碳酸盐缓冲液
    对于1×Dunn碳酸盐缓冲液,溶解NaHCO 3(10mM)0.84g/L,Na 2 CO 3 NO 3(3mM)0.318g/L和 甲醇(20%)200ml,在ddH 2 O 2中
  7. TBS
    在ddH 2 O中溶解Tris-HCl(50mM)6.05g/L和NaCl(150mM)8.76g/L并用HCl调节pH至7.5
  8. TBS-T
    补充有0.1%Tween20的TBS
  9. Western印迹缓冲液
    2%SDS
    62.5mM Tris-HCl(pH6.7) 100mMβ-巯基乙醇(仅在使用前直接添加)

致谢

这项工作由马普学会资助。

参考文献

  1. Chaudhury,A。和Howe,P.H。(2009)。 转化生长因子-β(TGFbeta)信号传导的故事:一种耻辱的谜。 IUBMB Life 61(10):929-939。
  2. Guasch,G.,Schober,M.,Pasolli,H.A.,Conn,E.B.,Polak,L。和Fuchs,E。(2007)。 TGFbeta信号丧失会破坏体内平衡并促进分层上皮细胞中的鳞状细胞癌。 Cancer Cell 12(4):313-327。
  3. Massague,J。(1998)。 TGF-β信号转导 Annu Rev Biochem 67 :753-791。
  4. Montanez,E.,Piwko-Czuchra,A.,Bauer,M.,Li,S.,Yurchenco,P.and Fassler,R。(2007)。 植入胚胎,造血系统和皮肤中的整合素功能的分析 < em> Methods Enzymol 426:239-289。
  5. Park,K.S.(2011)。 在胚胎干细胞中的Tgf-Beta家族信号传导。 Int J干细胞 4(1):18-23。
  6. Siegel,P.M。和Massague,J。(2003)。 TGF-β在动态平衡和癌症中的细胞凋亡和凋亡作用。 Rev Cancer 3(11):807-821
  7. Xiao,L.,Du,Y.,Shen,Y.,He,Y.,Zhao,H.and Li,Z.(2012)。 TGF-beta 1诱导的成纤维细胞增殖是由FGF-2/ERK通路介导的。 Front Biosci(Landmark Ed) 17:2667-2674。
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引用:Rognoni, E. (2014). TGFβ Stimulation Assay. Bio-protocol 4(23): e1313. DOI: 10.21769/BioProtoc.1313.
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