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TGFβ Release Co-culture Assay
TGFβ 释放共培养试验   

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Abstract

TGFβ is a potent cytokine modulating various processes including proliferation, differentiation, ECM synthesis and apoptosis (Siegel and Massague, 2003). Thus in many tissues availability of TGFβ is tightly regulated. TGFβ is secreted as an inactive complex where it is encapsulated by the latency associated protein (LAP), a ligand trap protein, which inhibits TGFβ binding to its receptor and retains TGFβ in the extracellular matrix (ten Dijke and Arthur, 2007). TGFβ can be released from the matrix and converted into its biological active form by huge number of processes including heat, high and low pH, release of reactive oxygen species (ROS) or various proteases (e.g. plasmin, elastase, matrix metalloproteinase-2 and -9) (Barcellos-Hoff and Dix, 1996; Lyons et al., 1988; Taipale et al., 1994; Yu and Stamenkovic, 2000). However, under physiological conditions the interaction of αv-class integrins with the RGD tripeptide motif in the LAP protein represents the key factor for TGFβ release in vivo. The relevance of integrin mediated TGFβ release for in vivo development and homeostasis is further underlined by the observation that mice with the integrin-binding deficient LAP proteins (RGD motif mutated to RGE) recapitulate all major phenotypes of TGFβ1 null mice, including multi-organ inflammation and defects in vasculogenesis (Shull et al., 1992; Yang et al., 2007). This striking phenotype overlap with TGFβ deficient mice and phenotypes of mice lacking αv-class integrins (Aluwihare et al., 2009; Bader et al., 1998) demonstrates an essential interconnection of integrins with TGFβ signaling in vivo, while the role of non-integrin mediated release mechanisms (ROS, pH, proteolytic cleavage etc.) during development remains less clear.
The TGFβ release assay measures the ability of cells to release TGFβ from a matrix. The assay was developed by (Annes et al., 2004) and we further optimized the protocol for keratinocytes. For other cell types the cell culture medium and culturing conditions would need to be adapted accordingly.
In keratinocytes TGFβ release is mainly mediated by αvβ6 integrin but also integrin αvβ3, αvβ5 and αvβ8 have been shown to liberate TGFβ, while other RGD binding integrins, such as α5β1 or α8β1 cannot release TGFβ (Asano et al., 2005a, 2005b; Mu et al., 2002; Munger et al., 1999). Mechanistically, the interaction with αvβ3, αvβ5 or αvβ6 integrin induces a conformational change in the LAP-TGFβ by generating an actin cytoskeleton dependent pulling force, allowing TGFβ to access its receptors. For αvβ8 integrin mediated TGFβ release it was shown that proteolytic cleavage is involved [see (Mu et al., 2002) for blocking conditions of TGFβ release by proteolytic cleavage and αvβ8 integrin].
The following protocol is optimized for the study of αvβ6-integrin mediated TGFβ release in keratinocytes.

Materials and Reagents

  1. Cell lines
    1. CHO-LTBP1 TGFβ rich matrix producing cell line, generated by the Daniel Rifkin lab expresses high levels of LTBP1-TGFβ (Annes et al., 2004).
    2. Transformed mink lung epithelial cells (tMLEC) TGFβ reporter cell line, stably expresses a luciferase reporter plasmid under control of a truncated plasminogen activator inhibitor type 1 promoter (PAI-1) (Abe et al., 1998).
      Note: CHO-LTBP1 and tMLEC are cultured in DMEM growth medium. tMLEC DMEM growth medium is supplemented with 250 mg/ml Geneticin (Life Technologies, InvitrogenTM, catalog number: 10131035 ).
  2. Antibodies
    1. αvβ6 integrin blocking antibody (Millipore, clone 10D5, catalog number: MAB2077Z )
    2. TGFβ neutralizing antibody (R&D Systems, clone 1D11, catalog number: MAB1835 )
  3. Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E6758 )
  4. BrightGlo luciferase assay kit (Promega Corporation, catalog number: E2610 )
  5. Dulbecco's minimum essential medium (DMEM) (Life Technologies, Gibco®, catalog number: 11966-025 )
  6. Fetal bovine serum (FBS) (Life Technologies, Gibco®, catalog number: 10270-106 )
  7. Calcium chloride (CaCl2) (Carl Roth, catalog number: A119.1 )
  8. Chelex 100 resin (Bio-Rad Laboratories, catalog number: 143-2832 )
  9. Penicillin-streptomycin (pen-strep) (Life Technologies, Gibco®, catalog number: 15070-063 )
  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: E9644 )
  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. L-Glutamine (Life Technologies, InvitrogenTM, catalog number: 25030-081 )
  18. Trypsin powder (Life Technologies, Gibco®, catalog number: 27250-018 )
  19.  0.5%Trypsin/EDTA (Life Technologies, Gibco®, catalog number: 15400-054 )
  20. Sodium chloride (NaCl) (Carl Roth, catalog number: P029 )
  21. di-Sodium hydrogen phosphate (Na2HPO4) (Carl Roth, catalog number: T876 )
  22. Hydrochloric acid (HCl) (Sigma-Aldrich, catalog number: 258148 )
  23. Potassium chloride (KCl) (Carl Roth, catalog number: HN02.3 )
  24. Potassium dihydrogen phosphate (KH2PO4) (Carl Roth, catalog number: 3904 )
  25. DMEM growth medium (see Recipes)
  26. Keratinocyte growth medium for murine keratinocyte culture (KGM) (see Recipes)
  27. Starving KGM (see Recipes)
  28. 0.4% keratinocyte trypsin (see Recipes)
  29. Phosphate-buffered saline (PBS) (see Recipes)
  30. PBS/EDTA (see Recipes)
  31. 0.1% trypsin/EDTA (see Recipes)
  32. Chelated FBS (see Recipes)

Equipment

  1. Flat bottom 96-well plates for cell culture (Corning, Costar®, catalog number: 3595 )
  2. Round bottom white 96-well plates (Corning, Costar®, catalog number: 3789 )
  3. 1.5 ml Eppendorf tubes (Sigma-Aldrich, catalog number: T9661-1000EA )
  4. Cell culture incubator (37 °C, 5% CO2)
  5. Standard centrifuge to spin down the cells
  6. Luminometer, e.g. GloMax (Promega corporation)
  7. Standard bright field microscope
  8. Multichannel pipette (recommended)

Procedure

  1. Considerations before you get started
    1. Consider carefully how many wells you need to coat with a TGFβ rich-matrix in order to perform all necessary control conditions, since matrix generation is the most time consuming step in this protocol. It is further recommended to prepare more wells, because it can happen at later steps that the matrix gets lost or CHO-LTBP1 cells cannot be removed properly in single wells.
    2. Cells which have been thawed should be passaged twice prior to the experiment.
    3. Each condition should be analyzed in triplicates.
    4. Conditions to include
      1. Only tMLEC → reporter cell back ground signal
      2. Cell of interest + tMLEC → untreated sample
      3. Cell of interest + tMLEC + αvβ6 blocking antibody → αvβ6 dependent TGFβ release control
      4. Cell of interest + tMLEC + TGFβ neutralizing antibody → TGFβ dependent luciferase reporter control. (This antibody blocks TGFβ binding to its high affinity TGFβ receptor.)
        Condition c can be extended or modified according to the TGFβ release mechanism which will be analyzed; e.g. for proteolytic and αvβ8 integrin-mediated release, see (Mu et al., 2002).
    5. During the assay there is no way to check for matrix loss as the matrix is difficult to see with bright field microscope. In parallel wells could be prepared where after cell removal the matrix is stained with antibodies against matrix components such as fibronectin and LTBP proteins.

  2. Preparation of a cell-free, TGFβ-rich matrix
    1. Plate 5.0 x 104 CHO-LTBP1 cells per well of a flat bottom 96-well plate in 100 µl DMEM growth medium and incubate in a cell culture incubator (37 °C, 5% CO2) for 48 h.
      Note: After 48 h the plated 96-well should be completely confluent, which can be checked under a standard bright field microscope. In the next steps, it is critical to remove the CHO-LTBP1 cells without destroying the TGFβ rich matrix and keeping the plate sterile. Thus each pipetting step should be performed very carefully without scratching the bottom of the wells. To avoid drying out of the wells the use of a multichannel pipette is recommended).
    2. Remove medium and add 100 µl PBS/EDTA solution on top of CHO-LTBP1 cells to wash off residual medium.
    3. Following the washing step add 100 µl PBS/EDTA and incubate for at least 30 min at 37 °C, 5% CO2.
      Note: Check with the microscope that cells round up and detach. If this is not the case prolong incubation for another 30 min.
    4. Remove the supernatant carefully with the multichannel pipette and add 100 µl PBS/EDTA. Gently pipette up and down 3-4 times and remove the supernatant. Repeat this step if necessary with 100 µl PBS/EDTA solution.
      Note: Check each well with the microscope in between washes to ensure complete removal of cells in all wells while avoiding too vigorous washing. If not all the cells are removed, remaining CHO-LTBP1 cells will re-adhere and generate/release TGFβ, contributing to the final result and thus generating false positive signals. On the other hand too harsh washing leads to a loss of the matrix. Further do not directly pipette on top of the matrix to avoid matrix damages. Thus it is recommended to tilt the plate by 45 °C during rinsing, so that the PBS/EDTA solution gently flows over the well bottom. Nevertheless, too many repeats of this gentle rinsing process can also cause matrix detachment.
    5. Then wash the matrix two times with PBS only at room temperature to remove all traces of EDTA.
      Note: Minimal traces of EDTA will impair keratinocyte adhesion and function! Thus this washing step is very important if you work with keratinocytes!
    6. Overlay the matrix coated wells with 50 µl starving KGM and keep at room temperature, while you prepare the cells, to avoid the drying of the matrix.

  3. Co-culture of the reporter cell line and cells of interest
    1. Detach cells of interest and TGFβ reporter cell line (tMLEC) from cell culture dishes. (For keratinocytes use keratinocyte trypsin, otherwise Trypsin/EDTA can be used). For trypsin digestion incubate cells with trypsin solution (37 °C, 5% CO2) and as soon as cells detach and rounded up stop the digestion and collect cells by adding complete medium. Then spin down single cell suspension (5 min, 900 rpm, 78 x g). It is important to wash suspended tMLEC cells once with starving KGM, to remove Ca2+ from the original DMEM medium.
    2. Per well mix 2.0 x 104 cells of interest (keratinocytes) and 1.5 x 104 tMLEC in 100 µl Starving KGM (total volume) in a 1.5 ml Eppendorf tube.
    3. For the antibody blocking controls (condition c and d) incubate mixed cells with αvβ6 integrin–blocking antibody (20 μg/ml) or TGFβ neutralizing antibody (15 μg/ml) in the 1.5 ml Eppendorf tube for 15 min at room temperature before plating.
    4. Remove starving KGM from matrix coated wells and quickly plate all conditions (100 µl total volume).
    5. Incubate 96-well plate for 16-24 h in a cell culture incubator (37 °C, 5% CO2).

  4. Detection of luciferase activity and data analysis
    Note: Procedure mainly follows the manufacturers’ protocol which can be downloaded on the Promega web site.
    1. Equilibrate BrightGlo reagent to room temperature and add 100 µl to each well after equilibrating the cell containing 96-well plate to room temperature for 5 min.
    2. Then pipet up and down 2-3 times to ensure efficient cell lysis, while avoiding air bubbles and transfer the complete content of each well (200 µl) to the white round bottom 96-well plate. (Before transfer efficient cell lysis can be quickly control under a bright field microscope.)
    3. Incubate for 5 min at room temperature before measuring luciferase activity with a luminometer, e.g. GloMax.
      Note: Before measuring the luciferase activity, make sure that all air bubbles have been removed, as they obscure signal detection.
    4. For data analysis average the triplicate measurements for each well and subtract the back ground signal (tMLEC only control).

Representative data



Figure 1. Luciferase activity of TGFβ reporter cell line (tMLEC) in TGFβ release co-culture assay with murine kerationcytes. Note the decrease in luciferase activity when cells are incubated with αvβ6 blocking antibody or TGFβ neutralizing antibody.

Recipes

  1. DMEM growth medium
    Dulbecco's minimum essential medium (DMEM) containing 10% heat-inactivated FBS and 1x pen-strep
  2. Keratinocyte growth medium for murine keratinocyte culture (KGM)
    Final working concentration
    Initial stock concentration
    Vol.
    MEM

    500 ml
    5 µg/ml insulin
    5 mg/ml in 4 mM 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
  3. 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 stored at 4 °C for up to 1 month
  4. 0.4% keratinocyte trypsin
    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
  5. 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
  6. PBS/EDTA
    PBS supplemented with 15 mM EDTA (should be sterile)
  7. Trypsin/EDTA
    Diluted 0.5% trypsin/EDTA with PBS to 0.1%
  8. 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 stored chelated FBS at -20 °C

Acknowledgments

The protocol is adapted from published work (Annes et al., 2004) and was funded by the Max Planck Society.

References

  1. Abe, M., Oda, N. and Sato, Y. (1998). Cell-associated activation of latent transforming growth factor-beta by calpain. J Cell Physiol 174(2): 186-193.
  2. Aluwihare, P., Mu, Z., Zhao, Z., Yu, D., Weinreb, P. H., Horan, G. S., Violette, S. M. and Munger, J. S. (2009). Mice that lack activity of alphavbeta6- and alphavbeta8-integrins reproduce the abnormalities of Tgfb1- and Tgfb3-null mice. J Cell Sci 122(Pt 2): 227-232.
  3. Annes, J. P., Chen, Y., Munger, J. S. and Rifkin, D. B. (2004). Integrin alphaVbeta6-mediated activation of latent TGF-beta requires the latent TGF-beta binding protein-1. J Cell Biol 165(5): 723-734.
  4. Asano, Y., Ihn, H., Yamane, K., Jinnin, M., Mimura, Y. and Tamaki, K. (2005a). Increased expression of integrin alpha(v)beta3 contributes to the establishment of autocrine TGF-beta signaling in scleroderma fibroblasts. J Immunol 175(11): 7708-7718.
  5. Asano, Y., Ihn, H., Yamane, K., Jinnin, M., Mimura, Y. and Tamaki, K. (2005). Involvement of alphavbeta5 integrin-mediated activation of latent transforming growth factor beta1 in autocrine transforming growth factor beta signaling in systemic sclerosis fibroblasts. Arthritis Rheum 52(9): 2897-2905.
  6. Bader, B. L., Rayburn, H., Crowley, D. and Hynes, R. O. (1998). Extensive vasculogenesis, angiogenesis, and organogenesis precede lethality in mice lacking all alpha v integrins. Cell 95(4): 507-519.
  7. Barcellos-Hoff, M. H. and Dix, T. A. (1996). Redox-mediated activation of latent transforming growth factor-beta 1. Mol Endocrinol 10(9): 1077-1083.
  8. Lyons, R. M., Keski-Oja, J. and Moses, H. L. (1988). Proteolytic activation of latent transforming growth factor-beta from fibroblast-conditioned medium. J Cell Biol 106(5): 1659-1665.
  9. Mu, D., Cambier, S., Fjellbirkeland, L., Baron, J. L., Munger, J. S., Kawakatsu, H., Sheppard, D., Broaddus, V. C. and Nishimura, S. L. (2002). The integrin alpha(v)beta8 mediates epithelial homeostasis through MT1-MMP-dependent activation of TGF-beta1. J Cell Biol 157(3): 493-507.
  10. Munger, J. S., Huang, X., Kawakatsu, H., Griffiths, M. J., Dalton, S. L., Wu, J., Pittet, J. F., Kaminski, N., Garat, C., Matthay, M. A., Rifkin, D. B. and Sheppard, D. (1999). The integrin alpha v beta 6 binds and activates latent TGF beta 1: a mechanism for regulating pulmonary inflammation and fibrosis. Cell 96(3): 319-328.
  11. Shull, M. M., Ormsby, I., Kier, A. B., Pawlowski, S., Diebold, R. J., Yin, M., Allen, R., Sidman, C., Proetzel, G., Calvin, D. and et al. (1992). Targeted disruption of the mouse transforming growth factor-beta 1 gene results in multifocal inflammatory disease. Nature 359(6397): 693-699.
  12. Taipale, J., Miyazono, K., Heldin, C. H. and Keski-Oja, J. (1994). Latent transforming growth factor-beta 1 associates to fibroblast extracellular matrix via latent TGF-beta binding protein. J Cell Biol 124(1-2): 171-181.
  13. ten Dijke, P. and Arthur, H. M. (2007). Extracellular control of TGFbeta signalling in vascular development and disease. Nat Rev Mol Cell Biol 8(11): 857-869.
  14. Yang, Z., Mu, Z., Dabovic, B., Jurukovski, V., Yu, D., Sung, J., Xiong, X. and Munger, J. S. (2007). Absence of integrin-mediated TGFbeta1 activation in vivo recapitulates the phenotype of TGFbeta1-null mice. J Cell Biol 176(6): 787-793.
  15. Yu, Q. and Stamenkovic, I. (2000). Cell surface-localized matrix metalloproteinase-9 proteolytically activates TGF-beta and promotes tumor invasion and angiogenesis. Genes Dev 14(2): 163-176.

简介

TGFβ是调节各种过程包括增殖,分化,ECM合成和凋亡的有效细胞因子(Siegel和Massague,2003)。因此,在许多组织中,TGFβ的可用性受到严格调控。 TGFβ作为无活性复合物分泌,其中其被潜伏相关蛋白(LAP)封闭,LAP是一种配体捕获蛋白,其抑制TGFβ与其受体结合并在细胞外基质中保留TGFβ(十Dijke和Arthur,2007)。 TGFβ可以从基质中释放并通过大量的过程包括热,高和低pH,活性氧(ROS)或各种蛋白酶(例如,纤溶酶,弹性蛋白酶)的释放而转化为其生物活性形式,基质金属蛋白酶-2和-9)(Barcellos-Hoff和Dix,1996; Lyons等人,1988; Taipale等人,1994; Yu和Stamenkovic, 2000)。然而,在生理条件下,αv-类整联蛋白与LAP蛋白中的RGD三肽基序的相互作用代表了体内TGFβ释放的关键因素。具有整联蛋白结合缺陷型LAP蛋白(RGD基序突变为RGE)的小鼠重现了TGFβ1缺失的所有主要表型,进一步强调了整合素介导的TGFβ释放对体内发育和体内平衡的相关性小鼠,包括多器官炎症和血管发生中的缺陷(Shull等人,1992; Yang等人,2007)。这种引人注目的表型与TGFβ缺陷小鼠重叠,缺少αv-类整联蛋白的小鼠的表型(Aluwihare等人,2009; Bader等人,1998)证明了重要的互连的整联蛋白与TGFβ信号传导的体内作用,而在开发期间非整联蛋白介导的释放机制(ROS,pH,蛋白水解切割等)的作用尚不清楚。
TGFβ释放测定测量细胞从基质释放TGFβ的能力。该测定由(Annes等人,2004)开发,并且我们进一步优化角质形成细胞的方案。对于其他细胞类型,细胞培养基和培养条件将需要相应地适应。在角质形成细胞中,TGFβ释放主要由αvβ6整联蛋白介导,但是整联蛋白αvβ3,αvβ5和αvβ8已经显示释放TGFβ,而其他RGD结合整联蛋白例如α5β1或α8β1不能释放TGFβ(Asano等人。,2005a,2005b; Mu ,2002; Munger等人,1999)。机械地,与αvβ3,αvβ5或αvβ6整联蛋白的相互作用通过产生肌动蛋白细胞骨架依赖性拉力而诱导LAP-TGFβ的构象变化,从而允许TGFβ接近其受体。对于αvβ8整联蛋白介导的TGFβ释放,表明涉及蛋白水解切割[参见(Mu等人,2002)用于通过蛋白水解切割和αvβ8整联蛋白阻断TGFβ释放的条件。
以下方案针对αvβ6整联蛋白介导的角质形成细胞中TGFβ释放的研究进行了优化。

材料和试剂

  1. 单元格行
    1. CHO-LTBP1富含TGFβ的基质产生细胞系,由Daniel产生 Rifkin实验室表达高水平的LTBP1-TGFβ(Annes等人,2004)。
    2. 转化貂肺上皮细胞(tMLEC)TGFβ报告细胞 线,在a的控制下稳定表达荧光素酶报告质粒 截短的纤溶酶原激活物抑制剂1型启动子(PAI-1)(Abe等人,1998)。
      注意:CHO-LTBP1和tMLEC在DMEM中培养 生长培养基。 tMLEC DMEM生长培养基补充有250mg/ml 遗传霉素(Life Technologies,Invitrogen TM,目录号:10131035)
  2. 抗体
    1. αvβ6整联蛋白阻断抗体(Millipore,clone 10D5,目录号:MAB2077Z)
    2. TGFβ中和抗体(R& D Systems,clone 1D11,目录号:MAB1835)
  3. 乙二胺四乙酸(EDTA)(Sigma-Aldrich,目录号:E6758)
  4. BrightGlo荧光素酶测定试剂盒(Promega Corporation,目录号:E2610)
  5. Dulbecco的最低必需培养基(DMEM)(Life Technologies,Gibco ,目录号:11966-025)
  6. 胎牛血清(FBS)(Life Technologies,Gibco ,目录号:10270-106)
  7. 氯化钙(CaCl 2)(Carl Roth,目录号:A119.1)
  8. Chelex 100树脂(Bio-Rad Laboratories,目录号:143-2832)
  9. 青霉素 - 链霉素(pen-strep)(Life Technologies,Gibco ,目录号:15070-063)
  10. 最小必需培养基(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:E9644)
  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. L-谷氨酰胺(Life Technologies,Invitrogen TM ,目录号:25030-081)
  18. 胰蛋白酶粉(Life Technologies,Gibco ,目录号:27250-018)
  19. < 0.5%胰蛋白酶/EDTA(Life Technologies,Gibco ,目录号:15400-054)
  20. 氯化钠(NaCl)(Carl Roth,目录号:P029)
  21. 磷酸氢二钠(Na 2 HPO 4)(Carl Roth,目录号:T876)
  22. 盐酸(HCl)(Sigma-Aldrich,目录号:258148)
  23. 氯化钾(KCl)(Carl Roth,目录号:HN02.3)
  24. 磷酸二氢钾(KH 2 PO 4)(Carl Roth,目录号:3904)
  25. DMEM生长培养基(参见配方)
  26. 用于小鼠角质形成细胞培养(KGM)的角质形成细胞生长培养基(参见Recipes)
  27. 饥饿KGM(参见食谱)
  28. 0.4%角质形成细胞胰蛋白酶(见配方)
  29. 磷酸盐缓冲盐水(PBS)(见配方)
  30. PBS/EDTA(参见配方)
  31. 0.1%胰蛋白酶/EDTA(见配方)
  32. 螯合FBS(参见配方)

设备

  1. 用于细胞培养的平底96孔板(Corning,Costar ,目录号:3595)
  2. 圆底白色96孔板(Corning,Costar ,目录号:3789)
  3. 1.5ml Eppendorf管(Sigma-Aldrich,目录号:T9661-1000EA)
  4. 细胞培养孵育器(37℃,5%CO 2)
  5. 标准离心机旋转下来细胞
  6. 照度计,例如GloMax(Promega公司)
  7. 标准明场显微镜
  8. 多通道移液器(推荐)

程序

  1. 开始之前的注意事项
    1. 仔细考虑需要用TGFβ包被多少孔 丰富矩阵,以便执行所有必要的控制条件,因为 矩阵生成是本协议中最耗时的步骤。 它 进一步推荐准备更多的井,因为它可以发生 后来的步骤,矩阵失去或CHO-LTBP1细胞不能 在单井中正确除去。
    2. 已经解冻的细胞应在实验前传代两次。
    3. 每个条件应分析三次。
    4. 包括的条件
      1. 只有tMLEC→报告单元背地信号
      2. 感兴趣的细胞+ tMLEC→未处理的样品
      3. 感兴趣的细胞+ tMLEC +αvβ6阻断抗体→αvβ6依赖性TGFβ释放控制
      4. 感兴趣的细胞+ tMLEC +TGFβ中和抗体→TGFβ 依赖性荧光素酶报告基因。 (该抗体阻断TGFβ 结合其高亲和性TGFβ受体。)
        →条件c可以 根据TGFβ释放机制延长或修饰 分析; 例如用于蛋白水解和αvβ8整联蛋白介导的释放, 参见(Mu等人,2002)。
    5. 在测定期间没有办法检查   对于矩阵损失,因为矩阵很难用亮场看到 显微镜。 在平行孔中可以在细胞去除后制备   用针对基质组分的抗体染色基质,   纤连蛋白和LTBP蛋白。

  2. 制备无细胞,富含TGFβ的基质
    1. 平板5.0×10 4个平底96孔板的每个孔中的CHO-LTBP1细胞 在100μlDMEM生长培养基中,并在细胞培养箱中孵育 (37℃,5%CO 2)中培养48小时。
      注意:48小时后,平板96孔 应完全融合,这可以在一个标准下检查 明场显微镜。在接下来的步骤中,关键是删除  CHO-LTBP1细胞,而不破坏富含TGFβ的基质并保持  板无菌。因此,每个移液步骤应该非常进行 小心不要划伤井底。避免干燥 推荐使用多通道移液管)。
    2. 除去培养基,并在CHO-LTBP1细胞顶部加入100μlPBS/EDTA溶液以洗去残余培养基。
    3. 在洗涤步骤后,加入100μlPBS/EDTA,并在37℃,5%CO 2下孵育至少30分钟。
      注意:用显微镜检查细胞向上和分离。如果不是这种情况延长孵育另外30分钟。
    4. 用多通道移液器小心地除去上清液 加入100μlPBS/EDTA。轻轻吸上下3-4次,删除  上清液。如有必要,用100μlPBS/EDTA重复此步骤 解决方案 注意:用显微镜检查每个孔 洗涤以确保完全去除所有孔中的细胞,同时避免 过于剧烈的洗涤。如果不是所有的单元都被移除,剩余 CHO-LTBP1细胞将重新粘附并产生/释放TGFβ,有贡献 到最终结果,从而产生假阳性信号。上的 其他手太苛刻的洗涤导致基质的损失。进一步 不直接吸取在矩阵的顶部,以避免基质损害。从而 建议在冲洗期间将板倾斜45°C,以便  PBS/EDTA溶液轻轻地流过孔底。不过,也 许多重复的这种温和的冲洗过程也可能导致基质 分离。
    5. 然后用PBS仅在室温下洗涤基质两次,以除去所有痕量的EDTA。
      注意:最小量的EDTA会损害角质形成细胞的粘附 功能! 因此,这个洗涤步骤是非常重要的,如果你使用 角质形成细胞!
    6. 用50μl覆盖基质涂覆的孔 饥饿KGM并保持在室温,而你准备细胞, 以避免基质的干燥

  3. 报道细胞系和感兴趣的细胞的共培养物
    1. 从细胞中分离目标细胞和TGFβ报道细胞系(tMLEC) 培养皿。 (对于角质形成细胞,使用角质形成细胞胰蛋白酶 可以使用胰蛋白酶/EDTA)。 对于胰蛋白酶消化孵育细胞 胰蛋白酶溶液(37℃,5%CO 2),并且一旦细胞脱离并且变圆   停止消化并通过加入完全培养基收集细胞。 然后   离心单细胞悬液(5分钟,900rpm,78×g)。 它是 重要的是用饥饿的KGM洗涤悬浮的tMLEC细胞一次 从原始DMEM培养基中除去Ca 2 +
    2. 每孔中混合2.0×10 4感兴趣的细胞(角质形成细胞)和1.5×10 4个tMLEC在100μl 将KGM(总体积)在1.5ml Eppendorf管中饥饿。
    3. 为了 抗体阻断对照(条件c和d)与混合细胞孵育   αvβ6整联蛋白阻断抗体(20μg/ml)或TGFβ中和 抗体(15μg/ml)在1.5ml Eppendorf管中在室温下15分钟 电镀前温度。
    4. 从基质涂层孔中除去饥饿的KGM,并快速平板所有条件(总体积100μl)
    5. 在细胞培养箱(37℃,5%CO 2)中孵育96孔板16-24小时。

  4. 检测荧光素酶活性和数据分析
    注意:过程主要遵循可以在Promega网站上下载的制造商协议。
    1. 平衡BrightGlo试剂到室温,并添加100微升到每个   在平衡含有96孔板的细胞至室后 温度5分钟。
    2. 然后用移液管上下吹2-3次 确保有效的细胞裂解,同时避免气泡和转移 将每个孔的完全内容物(200μl)加入到白色圆底 96孔板。 (转移前有效细胞裂解可快速 在明视场显微镜下进行控制。)
    3. 在室温下孵育5分钟,然后用发光计(例如GloMax)测量荧光素酶活性。
      注意:在测量荧光素酶活性之前,确保所有空气 气泡已被清除,因为它们遮挡了信号检测。
    4. 对于 数据分析平均每个孔的三次测量 减去背景信号(仅限tMLEC控制)。

代表数据



图1.TGFβ报道细胞系(tMLEC)在用鼠科动物细胞的TGFβ释放共培养测定中的荧光素酶活性。 请注意,当细胞与αvβ6封闭抗体或TGFβ中和抗体孵育时,荧光素酶活性降低。

食谱

  1. DMEM生长培养基
    含有10%热灭活的FBS和1x Pen-strep的Dulbecco最小必需培养基(DMEM)
  2. 用于小鼠角质形成细胞培养(KGM)的角质形成细胞生长培养基
    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+ +游离的) (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℃下储存最多1个月
  3. 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
  4. 0.4%角质形成细胞胰蛋白酶 将0.4g胰蛋白酶粉末溶解在100ml PBS中,通过0.2μm过滤器灭菌
    它可以在-20℃下储存1年,或者在+ 4℃下储存1个月。
    避免反复冻融周期
  5. 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,
  6. PBS/EDTA
    补充有15mM EDTA(应无菌)的PBS
  7. 胰蛋白酶/EDTA
    用PBS稀释0.5%胰蛋白酶/EDTA至0.1%
  8. Chelated FBS
    向FBS中加入水合Chelex 100树脂(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并在-20℃下储存螯合的FBS

致谢

该协议改编自出版的工作(Annes等人,2004),并且由马克斯普朗克学会资助。

参考文献

  1. Abe,M.,Oda,N。和Sato,Y。(1998)。 钙蛋白酶的潜在转化生长因子-β的细胞相关激活。 J Cell Physiol 174(2):186-193。
  2. Aluwihare,P.,Mu,Z.,Zhao,Z.,Yu,D.,Weinreb,P.H.,Horan,G.S.,Violette,S.M.and Munger,J.S。(2009)。 缺乏活动的小鼠 αvβ6和αvβ-整联蛋白再现了Tgfb1-和Tgfb3-null小鼠的异常。 J Cell Sci 122(Pt 2):227-232。
  3. Annes,J.P.,Chen,Y.,Munger,J.S。和Rifkin,D.B。(2004)。 整合素αVβ6介导的休眠TGF-β活化需要潜在的TGF-β结合蛋白-1。 J Cell Biol 165(5):723-734。
  4. Asano,Y.,Ihn,H.,Yamane,K.,Jinnin,M.,Mimura,Y.and Tamaki,K。(2005a)。 整合素α(v)β3的增加的表达有助于在硬皮病中建立自分泌TGF-β信号传导成纤维细胞。 J Immunol 175(11):7708-7718。
  5. Asano,Y.,Ihn,H.,Yamane,K.,Jinnin,M.,Mimura,Y.and Tamaki,K。(2005)。 参与αvβ5整联蛋白介导的潜在转化生长因子β1在自分泌转化生长因子β信号传导中的活化系统性硬化症成纤维细胞。关节炎风湿(Rheum) 52(9):2897-2905。
  6. Bader,B.L.,Rayburn,H.,Crowley,D。和Hynes,R.O。(1998)。 在缺乏所有αv整联蛋白的小鼠中,广泛的血管生成,血管生成和器官发生早于致死性。 Cell 95(4):507-519。
  7. Barcellos-Hoff,M.H。和Dix,T.A。(1996)。 氧化还原介导的潜伏转化生长因子-β1的激活。 Endocrinol 10(9):1077-1083。
  8. Lyons,R.M.,Keski-Oja,J.and Moses,H.L。(1988)。 从成纤维细胞条件培养基中潜在转化生长因子-β的蛋白水解活化。 J Cell Biol 106(5):1659-1665。
  9. Mu,D.,Cambier,S.,Fjellbirkeland,L.,Baron,J.L.,Munger,J.S.,Kawakatsu,H.,Sheppard,D.,Broaddus,V.C。和Nishimura, 整合素α(v)beta8通过TGF-β1的MT1-MMP依赖性激活介导上皮内环境稳态。 J Cell Biol 157(3):493-507。
  10. Munger,JS,Huang,X.,Kawakatsu,H.,Griffiths,MJ,Dalton,SL,Wu,J.,Pittet,JF,Kaminski,N.,Garat,C.,Matthay,MA,Rifkin,DB和Sheppard ,D.(1999)。 整合素αvβ6结合并激活潜伏的TGF beta 1:调节肺部炎症的机制fibrosis。 Cell 96(3):319-328。
  11. Shull,M.M.,Ormsby,I.,Kier,A.B.,Pawlowski,S.,Diebold,R.J.,Yin,M.,Allen,R.,Sidman,C.,Proetzel,G.,Calvin,D.and et al。 (1992)。 小鼠转化生长因子-β1基因的靶向破坏导致多灶性炎症性疾病。 359(6397):693-699。
  12. Taipale,J.,Miyazono,K.,Heldin,C.H。和Keski-Oja,J。(1994)。 潜在转化生长因子-β1通过潜在TGF-β结合蛋白与成纤维细胞胞外基质结合。/a> J Cell Biol 124(1-2):171-181。
  13. ten Dijke,P。和Arthur,H.M。(2007)。 TGFbeta信号在血管发育和疾病中的细胞外控制。 Nat Rev Mol Cell Biol 8(11):857-869。
  14. Yang,Z.,Mu,Z.,Dabovic,B.,Jurukovski,V.,Yu,D.,Sung,J.,Xiong,X.and Munger,J.S。(2007)。 缺乏整合素介导的TGFbeta1活化体内重现TGFbeta1的表型-null mice。 J Cell Biol 176(6):787-793。
  15. Yu,Q.and Stamenkovic,I。(2000)。 细胞表面定位的基质金属蛋白酶-9蛋白水解活化TGF-β并促进肿瘤侵袭和血管生成。/a> Dev 14(2):163-176。
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引用:Rognoni, E. (2014). TGFβ Release Co-culture Assay. Bio-protocol 4(23): e1314. DOI: 10.21769/BioProtoc.1314.
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