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Oct 2019

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In-vitro GLP-1 Release Assay Using STC-1 Cells
STC-1细胞体外释放GLP-1的实验研究   

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Abstract

Enteroendocrine cells (EECs) are known chemosensors in the gastrointestinal (GI) epithelium. They release a diversity of gut hormones in response to various stimuli. Here, we report an in-vitro assay to measure GLP-1 release from cultured murine EEC’s under fatty acid stimulation.

Keywords: Enteroendocrine cells (肠内分泌细胞), L-cells (L细胞), STC-1 (STC-1细胞), Docosahexaenoic acid (DHA) (二十二碳六烯酸), Glucagon-like peptide-1 (GLP-1) (胰高血糖素样肽-1)

Background

The intestinal epithelium is the largest interface between the human body and the environment. This interface consists of a single epithelial layer containing several distinct cell-types with unique functional roles. Differentiated from LGR5-positive epithelial stem cells, enteroendocrine cells (EECs) are scattered throughout the epithelium along the entire GI tract. EECs play a critical role in responses to nutrient and other stimuli by producing and secreting a variety of hormones. Traditionally, these cells are classified by their hormonal profile (Nausheen et al., 2013; Latorre et al., 2016; Verhoeckx et al., 2015a), although with the advent of single cell RNA-sequencing, we have come to appreciate that most of these cells secrete multiple hormones (Haber et al., 2017; Billing et al., 2019). A subtype of EECs, L-cells, are defined by the production of glucagon-like peptides 1 and 2 (GLP-1 and GLP-2), and may also secrete peptide YY (PYY) and insulin-like peptide 5 (INSL5). Most L-cells are found in the distal ileum and colon, while some are also found proximally in the duodenum and jejunum. The receptors on the surface of L-cells allow them to directly sense luminal components and produce appropriate hormone responses. The most notable receptors of nutrients and food components on L-cells are the classic taste receptors, free fatty acid receptors and receptors for protein and their products (Drucker and Nauck, 2006; Furness et al., 2013; Spreckley and Murphy, 2015; Gribble and Reimann, 2016; Parada Venegas et al., 2019). L-cells can also sense LPS through expressing Toll-like receptors (TLRs) (Lebrun et al., 2017). By producing hormones, L-cells are able to stimulate carbohydrate uptake and insulin release, slow down intestinal transit and regulate appetite.

To study the role of L cells in-vitro, numerous human and rodent mouse cell lines have been derived. The NCI-H716 cell line, the only human EEC line, was derived from a spontaneous colorectal tumor. The NCI-H716 cell line produces GLP-1, PYY and mucin and is thought to represent distal L-cells. They require a basement membrane to attach to culture plates and only 30% of NCI-H716 cells produce PYY, thus limiting their use in routine in-vitro experiments (Park et al., 1987; Verhoeckx et al., 2015b). To overcome the limitations posed by this cell line, non-human EECs have been derived. Notably, GLUTag and STC-1 are of great utility in the study of GLP-1 secretion. The GLUTag cell line is a single-cell subcloned population of L-cells, established from a large bowel tumor in proglucagon-SV40 Large T antigen transgenic mice. They are easy to transfect and secrete GLP-1 and CCK. Like the NIC-H716 cells, GLUTag cells also need attachment matrices (Chang et al., 1994; Drucker et al., 1994; Verhoeckx et al., 2015b). In contrast to GLUTag cells, the STC-1 cell line was established from a secretin producing tumor, which arose in the duodenum of double transgenic mice expressing proinsulin-SV40 Large T antigen/proinsulin-polyoma x Small T antigen. Like native intestinal L-cells, STC-1 cells produce GLP-1, CCK, GIP, PYY, pancreatic polypeptide, neurotensin, GLP-2 and oxyntomodulin, in varying amounts (Rindi et al., 1990; Verhoeckx et al., 2015b). They are easy to culture without the need for an attachment matrix. These features make STC-1 cells a good model to study hormonal secretion mechanisms.

Like EECs of the large intestine, STC-1 cells harbor a variety of nutrient receptors. Prior work has demonstrated that various substances binding to specific cell-surface receptors on STC-1 cells results in hormone secretion (McCarthy et al., 2015). Notably, fatty acids elicit strong CCK secretion from STC-1 cells, thus they have been extensively used to study CCK secretion dynamics. However, few studies have focused on fatty acid induced GLP-1 secretion or have failed to address variation in cellular density (McLaughlin et al., 1998; Sidhu et al., 2000; Benson et al., 2002; Hirasawa et al., 2005). Here we describe a method to measure GLP-1 secretion from STC-1 cells upon fatty acid stimulation.

Materials and Reagents

  1. 10 ml Glass bottles
  2. 50 ml Glass beaker
  3. 5 ml Glass pipettes (Corning, catalog number: 7077-5N )
  4. Capillary Micropipettes (Drummond Scientific Company, catalog number: 5-000-2005 )
  5. 6-cm dishes (Corning, catalog number: 430166 )
  6. 10 ml and 50 ml sterile plastic pipettes with filter (Corning, catalog numbers: 4488 , 4490 )
  7. Sterile cell lifts (Corning, catalog number: 3008 )
  8. 1.5 ml microcentrifuge tubes (Fisherbrand, catalog number: 05-408-129 )
  9. 1.5 ml disposable cuvettes (BRAND, catalog number: 759076D )
  10. STC-1 cell line (ATCC, catalog number: CRL-3254TM)
  11. Phosphate-buffered saline (Sigma, catalog number: D8537 )
  12. Trypsin (Corning, catalog number: 25-053-CI )
  13. DMEM, High Glucose (Hyclone, catalog number: SH30022.02 )
  14. Fetal Bovine Serum (Gibco, catalog number: 35050-061 )
  15. Enrofloxacin (Sigma, catalog number: 17849-5G-F )
  16. Docosahexaenoic acid, DHA (Nu-chek-policy, U-84-A DHA, 1GM), DHA comes as a solution
  17. Pure ethanol (Pharmco Products, catalog number: 111000200C1GL )
  18. Nitrogen gas
  19. HEPES (Sigma, catalog number: H3375 )
  20. NaCl (Fisher Scientific, catalog number: BP358-10 )
  21. KCl (Sigma, catalog number: P9333 )
  22. CaCl2 (Sigma, catalog number: C1016 )
  23. MgCl2 (Sigma, catalog number: M8266 )
  24. EDTA (OmniPur, catalog number: 4050 )
  25. Triton X-100 (Fisher Scientific, catalog number: BP151-500 )
  26. Tris-base (Fisher Scientific, catalog number: BP152-5 )
  27.  PMSF (MP biomedicals, catalog number: 195381 )
  28. HaltTM Protease and Phosphatase Inhibitor Cocktail (100x) (Thermo Fisher Scientific, catalog number: 78444 )
  29. Sterile water
  30. Protein assay dye reagent concentrate (Bio-Rad, catalog number: 5000006 )
  31. High sensitive glugagon-like peptide(GLP-1) active chemiluminescent ELISA kit (Millipore, catalog number: EZGLPHS-35K )
  32. 10 mg/ml DHA stock solution (see Recipes)
  33. HEPES buffer (see Recipes)
  34. 100 µM DHA Stimulation buffer (see Recipes)
  35. Cell lysis buffer (see Recipes)
  36. 100 mM PMSF stock solution (see Recipes)

Equipment

  1. Portable Pipet-Aid® XP Pipette Controller (Drummond Scientific Company, catalog number: 4-000-101 )
  2. Tissue culture incubator
  3. Refrigerated microcentrifuge (Eppendorf, model: 5424R )
  4. Sonicator (Branson 250 Sonifier)
  5. ELISA plate reader (TECAN, Sunrise absorbance microplate reader)

Procedure

  1. Prepare 10 mg/ml docosahexaenoic acid (DHA) stock solution (see Recipe)
    1.  Dilute DHA solution with pure ethanol at 10 mg/ml.
      Note: DHA will stick to plastic material. Always remember to use glass pipettes when you work with fatty acids.
    2. Aliquot DHA stock solution into small glass bottles, 10 ml each.
    3. Ambient air above the solution should be displaced to avoid oxidation of DHA by priming pure nitrogen gas on the surface of the solution prior to closing the container.
    4. Store at -20 °C.

  2.  DHA Stimulation assay
    1. Seed 2 x 106 STC-1 cells in 6-cm dishes two days prior to experiment in duplicates. Culture the cells in tissue culture incubator at 37 °C, 5% CO2, using DMEM with 10% FBS and 20 ng/ml enrofloxacin.
    2. On experimental day cells should be 80% confluent.
    3. Aspirate media and wash the cells twice with 3 ml of HEPES buffer.
    4. Add 3 ml of HEPES buffer into each dish.
    5. Put cells back to tissue incubator for 30 min. The lack of nutrients in the buffer is meant to serve as a ‘starvation’ period prior to nutrient stimulation.
    6. Prepare 100 µM DHA stimulation buffer.
      1. To prepare 20 ml of 100 µM DHA stimulation buffer, add 66 µl of 10 mg/ml DHA (30.4 mM) stock solution into a glass beaker using glass capillary micropipettes. Then add 20 ml of HEPES buffer.
      2. Sonicate the solution immediately at output level 5 and constant duty cycle for 3 min prior to stimulation to prevent DHA precipitation. If using a different sonicator, set the amplitude as 40%. After sonication, the solution should be clear, with minimal bubbling.
        Note: Always prepare this DHA solution freshly, just prior to use.
    7. Aspirate HEPES buffer gently. Then add 1 ml of HEPES buffer into each control dish and 1 ml of DHA stimulation buffer into each stimulation dish. Put all dishes back to tissue culture incubator for 15 min.
    8. Collect 600 µl of media from each dish and put into a fresh 1.5 ml microcentrifuge tube, then place them on ice.
    9. Add 0.6 µl of 100 mM PMSF (see Recipe) into the 600 µl of media at final concentration of 100 µM.
    10. Spin the microcentrifuge tubes at 850 x g for 5 min, at 4 °C.
    11. Transfer 500 µl of media into a fresh tube.
      Note: Samples are now ready for ELISA measurement. One can also store samples at -80 °C up to two weeks.
    12. Measure GLP-1 concentration in samples by ELISA kit according to manufacturer’s instructions. Make sure to set the standard as suggested by the manufacturer.

  3. Lyse the cells in the culture and measure protein concentration from all dishes by Bradford assay   Note: This is meant to account for differences in the number of cells in each dish in these experiments.
    1. Carefully remove any residual HEPES buffer in each dish.
    2. Add 1 ml of cold PBS into each dish.
    3. Scrape cells down very carefully using a cell lift. 
    4. Collect cells into a 1.5 ml microcentrifuge tube and put on ice.
    5.  Spin the tubes at 850 x g for 5 min, at 4 °C.
    6. Aspirate supernatants, then add 50 µl of cell lysis buffer and pipette up and down gently to homogenize. Put tubes on ice.
    7. Lyse the cells for 20 min. Vortex every 5 min. 
    8. Spin the tubes at 21,130 x g for 15 min, at 4 °C.
    9. Transfer supernatants into a fresh tube.
    10.  Measure protein concentration using protein assay dye reagent concentrate according to manufacturer’s instructions.

Data analysis

Calculate measured GLP-1 concentration of each sample appropriately from three independent experiments, duplicates in each experiment. Then normalize the value to its corresponding protein concentration value as measured by Bradford assay (Table 1). Use any software package with 4-parameter logistic regression for ELISA analysis.


Table 1. Adjusted GLP-1 concentration


Notes

Using healthy cells is critical for experiments to work. One should check cell growth status and mycoplasma routinely. Always remember to fill DHA stock solution with nitrogen and prepare stimulation buffer fresh prior to use. If one freezes samples at -80 °C, do not freeze and thaw repeatedly.

Recipes

  1. 10 mg/ml DHA stock solution
    1. 600 µl of DHA liquid coming from the manufacturer (1 g, 1.2 ml in total)
    2. 49.4 ml of 100% pure ethanol
    3. Aliquot into small glass bottles and fill with nitrogen gas. Store at -20 °C
  2. HEPES buffer
    20 mM HEPES, pH 7.9
    140 mM NaCl
    4.5 mM KCl
    1.2 mM CaCl2
    1.2 mM MgCl2
  3. 100 µM DHA Stimulation buffer
    66 µl of 10 mg/ml DHA stock solution
    20 ml of HEPES buffer
    Sonicate the solution at output level 5 constant duty cycle for 3 min prior to use
  4.  Cell lysis buffer
    25 mM HEPES, pH 7.9
    100 mM NaCl
    1 mM EDTA
    10% Glycerol
    1% Triton X-100
  5. 100 mM PMSF stock solution
    174.2 mg PMSF
    10 ml of 100% ethanol

Acknowledgements

This work was made possible by funding to LS-D from the following sources: NIH 5 K12 HD-068369–05 and Children’s Health Clinical Research Advisory Committee: CCRAC 195.
  This protocol was adapted from Hirasawa et al. (2005).

Competing interests

The authors declare no competing interests.

References

  1. Benson, R. S., Sidhu, S., Jones, M. N., Case, R. M. and Thompson, D. G. (2002). Fatty acid signalling in a mouse enteroendocrine cell line involves fatty acid aggregates rather than free fatty acids. J Physiol 538(Pt 1): 121-131. 
  2. Billing, L. J., Larraufie, P., Lewis, J., Leiter, A., Li, J., Lam, B., Yeo, G. S., Goldspink, D. A., Kay, R. G., Gribble, F. M. and Reimann, F. (2019). Single cell transcriptomic profiling of large intestinal enteroendocrine cells in mice - Identification of selective stimuli for insulin-like peptide-5 and glucagon-like peptide-1 co-expressing cells. Mol Metab 29: 158-169.
  3. Chang, C. H., Chey, W. Y., Sun, Q., Leiter, A. and Chang, T. M. (1994). Characterization of the release of cholecystokinin from a murine neuroendocrine tumor cell line, STC-1. Biochim Biophys Acta 1221(3): 339-347.
  4. Drucker, D. J., Jin, T., Asa, S. L., Young, T. A. and Brubaker, P. L. (1994). Activation of proglucagon gene transcription by protein kinase-A in a novel mouse enteroendocrine cell line. Mol Endocrinol 8(12): 1646-1655.
  5. Drucker, D. J. and Nauck, M. A. (2006). The incretin system: glucagon-like peptide-1 receptor agonists and dipeptidyl peptidase-4 inhibitors in type 2 diabetes. Lancet 368(9548): 1696-1705.
  6. Furness, J. B., Rivera, L. R., Cho, H. J., Bravo, D. M. and Callaghan, B. (2013). The gut as a sensory organ. Nat Rev Gastroenterol Hepatol 10(12): 729-740.
  7. Gribble, F. M. and Reimann, F. (2016). Enteroendocrine Cells: Chemosensors in the Intestinal Epithelium. Annu Rev Physiol 78: 277-299.
  8. Haber, A. L., Biton, M., Rogel, N., Herbst, R. H., Shekhar, K., Smillie, C., Burgin, G., Delorey, T. M., Howitt, M. R., Katz, Y., Tirosh, I., Beyaz, S., Dionne, D., Zhang, M., Raychowdhury, R., Garrett, W. S., Rozenblatt-Rosen, O., Shi, H. N., Yilmaz, O., Xavier, R. J. and Regev, A. (2017). A single-cell survey of the small intestinal epithelium. Nature 551(7680): 333-339.
  9. Hirasawa, A., Tsumaya, K., Awaji, T., Katsuma, S., Adachi, T., Yamada, M., Sugimoto, Y., Miyazaki, S. and Tsujimoto, G. (2005). Free fatty acids regulate gut incretin glucagon-like peptide-1 secretion through GPR120. Nat Med 11(1): 90-94. 
  10.  Latorre, R., Sternini, C., De Giorgio, R. and Greenwood-Van Meerveld, B. (2016). Enteroendocrine cells: a review of their role in brain-gut communication. Neurogastroenterol Motil 28(5): 620-630. 
  11. Lebrun, L. J., Lenaerts, K., Kiers, D., Pais de Barros, J. P., Le Guern, N., Plesnik, J., Thomas, C., Bourgeois, T., Dejong, C. H. C., Kox, M., Hundscheid, I. H. R., Khan, N. A., Mandard, S., Deckert, V., Pickkers, P., Drucker, D. J., Lagrost, L. and Grober, J. (2017). Enteroendocrine L cells sense LPS after gut barrier injury to enhance GLP-1 secretion. Cell Rep 21(5): 1160-1168.
  12. McCarthy, T., Green, B. D., Calderwood, D., Gillespie, A., Cryan, J. F. and Giblin, L. (2015). STC-1 Cells. In: Verhoeckx, K., Cotter, P., López-Expósito, I., Kleiveland, C., Lea, T., Mackie, A., Requena, T., Swiatecka, D. and Wichers, H. (Eds.). In: The Impact of Food Bioactives on Health. Springer, 211-220.
  13. McLaughlin, J. T., Lomax, R. B., Hall, L., Dockray, G. J., Thompson, D. G. and Warhurst, G. (1998). Fatty acids stimulate cholecystokinin secretion via an acyl chain length-specific, Ca2+-dependent mechanism in the enteroendocrine cell line STC-1. J Physiol 513 (Pt 1): 11-18. 
  14. Nausheen, S., Shah, I. H., Pezeshki, A., Sigalet, D. L. and Chelikani, P. K. (2013). Effects of sleeve gastrectomy and ileal transposition, alone and in combination, on food intake, body weight, gut hormones, and glucose metabolism in rats. Am J Physiol Endocrinol Metab 305(4): E507-518.
  15. Parada Venegas, D., De la Fuente, M. K., Landskron, G., Gonzalez, M. J., Quera, R., Dijkstra, G., Harmsen, H. J. M., Faber, K. N. and Hermoso, M. A. (2019). Short chain fatty acids (SCFAs)-mediated gut epithelial and immune regulation and its relevance for inflammatory bowel diseases. Front Immunol 10: 277. 
  16. Park, J. G., Kramer, B. S., Steinberg, S. M., Carmichael, J., Collins, J. M., Minna, J. D. and Gazdar, A. F. (1987). Chemosensitivity testing of human colorectal carcinoma cell lines using a tetrazolium-based colorimetric assay. Cancer Res 47(22): 5875-5879.
  17.  Rindi, G., Grant, S. G., Yiangou, Y., Ghatei, M. A., Bloom, S. R., Bautch, V. L., Solcia, E. and Polak, J. M. (1990). Development of neuroendocrine tumors in the gastrointestinal tract of transgenic mice. Heterogeneity of hormone expression. Am J Pathol 136(6): 1349-1363.
  18. Sidhu, S. S., Thompson, D. G., Warhurst, G., Case, R. M. and Benson, R. S. (2000). Fatty acid-induced cholecystokinin secretion and changes in intracellular Ca2+ in two enteroendocrine cell lines, STC-1 and GLUTag. J Physiol 528 Pt 1: 165-176.
  19. Spreckley, E. and Murphy, K. G. (2015). The L-Cell in nutritional sensing and the regulation of appetite. Front Nutr 2: 23. 
  20. Verhoeckx, K., Cotter, P., López-Expósito, I., Kleiveland, C., Lea, T., Mackie, A., Requena, T., Swiatecka, D. and Wichers, H. Eds (2015a). General Introduction. In: The Impact of Food Bioactives on Health: In Vitro and Ex Vivo Models VII-XIII. Springer.
  21. Verhoeckx, K., Cotter, P., López-Expósito, I., Kleiveland, C., Lea, T., Mackie, A., Requena, T., Swiatecka, D. and Wichers, H. Eds (2015b). Part IV, Enteroendocrine Cell Models: General Introduction. In: The Impact of Food Bioactives on Health: In Vitro and Ex Vivo Models. Springer.

简介

[摘要] 肠内分泌细胞(EEC)是胃肠道(GI)上皮中的已知化学传感器。它们响应各种刺激而释放多种肠激素。在这里,我们报告了一种体外测定法,以测量在脂肪酸刺激下培养的鼠EEC中GLP-1的释放。

[背景] 肠上皮是人体与环境之间最大的界面。该界面由单个上皮层组成,包含多个具有独特功能角色的不同细胞类型。与LGR5阳性上皮干细胞不同,肠内分泌细胞(EEC)沿整个胃肠道散布在整个上皮中。EEC通过产生和分泌多种激素,在对营养和其他刺激的反应中起关键作用。传统上,这些细胞是按照荷尔蒙特征来分类的(Nausheen 等人,2013; Latorre 等人,2016; Verhoeckx 等人,2015a),尽管随着单细胞RNA测序的到来,我们逐渐意识到这些细胞中的大多数分泌多种激素(Haber 等人,2017; Billing 等人,2019)。EEC的亚型L细胞由胰高血糖素样肽1和2(GLP-1和GLP-2)的产生定义,也可能分泌YY肽(PYY)和胰岛素样肽5(INSL5) 。大多数L细胞见于回肠末端和结肠,而另一些也见于十二指肠和空肠近端。L细胞表面的受体使它们能够直接感知管腔成分并产生适当的激素反应。L细胞上最显着的营养成分和食物成分受体是经典的味觉受体,游离脂肪酸受体以及蛋白质及其产物的受体(Drucker 和Nauck ,2006年;Furness 等,2013年; Spreckley 和Murphy ,2015年。Gribble 和Reimann ,2016; Parada Venegas 等,2019)。L细胞还可以通过表达Toll样受体(TLR)来感知LPS (Lebrun 等,2009 )。通过产生激素,L细胞能够刺激碳水化合物的摄取和胰岛素的释放,减慢肠道的运输并调节食欲。

为了研究L细胞的体外作用,已经获得了许多人类和啮齿动物的小鼠细胞系。NCI-H716细胞系是唯一的人EEC系,来自自发性结直肠肿瘤。NCI-H716细胞系产生GLP-1,PYY和粘蛋白,并被认为代表远端L细胞。它们需要基底膜附着在培养板上,只有30%的NCI-H716细胞产生PYY,因此限制了它们在常规体外实验中的使用(Park 等人,1987; Verhoeckx 等人,2015b)。为了克服该细胞系带来的限制,已经衍生出非人类EEC。值得注意的是,GLUTag 和STC-1在研究GLP-1分泌方面非常有用。所述的GLUTag 细胞系是一种单细胞亚克隆L-细胞群,在高血糖素原SV40大T抗原转基因小鼠从大肠肿瘤建立的。它们易于转染并分泌GLP-1和CCK。像NIC-H716细胞一样,GLUTag 细胞也需要附着基质(Chang 等,1994; Drucker 等,1994; Verhoeckx 等,2015b)。在相反的GLUTag 细胞中,STC-1细胞系成立来回m是一促胰液产生肿瘤,其产生于双转基因小鼠的十二指肠中表达胰岛素原-SV40大T抗原/胰岛素原多瘤X小T抗原。像天然肠道L细胞一样,STC-1细胞产生不同数量的GLP-1,CCK,GIP,PYY,胰多肽,神经降压素,GLP-2和胃泌酸调节素(Rindi 等人,1990; Verhoeckx 等人,2015b )。它们易于培养,无需附着基质。这些特征使STC-1细胞成为研究激素分泌机制的良好模型。

像大肠的EEC一样,STC-1细胞也具有多种营养受体。先前的工作表明,与STC-1细胞上特定细胞表面受体结合的各种物质都会导致激素分泌(McCarthy 等,2015)。值得注意的是,脂肪酸引起STC-1细胞强烈的CCK分泌,因此它们已被广泛用于研究CCK分泌动力学。但是,很少有研究专注于脂肪酸诱导的GLP-1分泌或未能解决细胞密度的变化(McLaughlin 等,1998; Sidhu 等,2000;Benson 等,2002;Hirasawa 等, 2005年)。在这里,我们描述了一种在脂肪酸刺激下测量STC-1细胞GLP-1分泌的方法。

关键字:肠内分泌细胞, L细胞, STC-1细胞, 二十二碳六烯酸, 胰高血糖素样肽-1

材料和试剂


 


10毫升玻璃瓶
50毫升玻璃烧杯
500万升玻璃移液器(Corning,目录号:7077-5N)
毛细管微量移液器(Drummond Scientific Company,目录号:5-000-2005)
6厘米长的盘子(Corning,货号:430166)
10 ml和50 ml带过滤器的无菌塑料移液器(Corning,目录号s :4488、4490)
无菌细胞移位机(Corning,目录号:3008)
1.5 ml微量离心管(Fisherbrand ,目录号05-408-129)
1.5毫升一次性比色皿(品牌,目录号:759076D)
STC-1细胞系(ATCC,目录号:CRL-3254 TM )
磷酸盐缓冲盐水(Sigma,目录号:D8537)
胰蛋白酶(Corning,目录号:25-053-CI)
DMEM,高葡萄糖(Hyclone ,目录号:SH30022.02)
胎牛血清(Gibco,目录号:35050-061)
恩诺沙星(Sigma,目录号:17849-5G-F)
二十二碳六烯酸DHA(Nu-chek-policy,U-84-A DHA,1GM ),DHA作为解决方案
纯乙醇(Pharmco 产品,目录号:111000200C1GL)
氮气
HEPES(Sigma,目录号:H3375)
NaCl(Fisher Scientific,目录号BP358-10)
氯化钾(Sigma,目录号:P9333)
CaCl 2 (Sigma,目录号:C1016)
MgCl 2 (Sigma,目录号:M8266)
EDTA(OmniPur ,目录号:4050)
海卫一X-100(Fisher Scientific,目录号:BP151-500)
Tris-base(Fisher Scientific,目录号:BP152-5)
PMSF(MP生物医学,目录号:195381)
Halt TM 蛋白酶和磷酸酶抑制剂混合物(100x )(Thermo Fisher Scientific,目录号:78444)
无菌水
蛋白质测定染料试剂浓缩液(Bio-Rad,目录号:5000006)
高灵敏度类胰高血糖素样肽(GLP-1)活性化学发光ELISA试剂盒(Millipore,目录号:EZGLPHS-35K)
10 mg / ml DHA储备溶液(请参阅食谱)
HEPES缓冲区(请参阅食谱)
100 DM DHA刺激缓冲液(请参阅食谱)
细胞裂解缓冲液(请参阅食谱)
100 mM PMSF储备溶液(请参阅配方)
 


设备


 


便携式吸取急救® XP移液器控制器(德拉蒙德小号系统求解Ç ompany,目录号:4-000-101)
组织培养培养箱
冷冻微量离心机(Eppendorf ,型号:5424R)
超声波仪(Branson 250 Sonifier )
ELISA酶标仪(TECAN,日出吸光度酶标仪)
 


程序


 


准备10 mg / ml二十二碳六烯酸(DHA)储备溶液(请参阅配方)
用纯乙醇以10 mg / ml 稀释D HA溶液。
注意:DHA会粘在塑料上。处理脂肪酸时,请务必记住使用玻璃移液器。


等分试样DHA储备液分成小玻璃瓶10 米升每。
溶液上方的环境空气应排空,以避免DHA氧化,方法是在关闭容器之前在溶液表面上灌注纯氮气,以注入纯净的氮气。
储存在-20°C。
 


DHA刺激测定
实验前两天,将2 x 10 6 STC-1细胞接种在6厘米长的培养皿中,一式两份。使用含10%FBS和20 ng / ml恩诺沙星的DMEM 在37°C,5%CO 2的组织培养培养箱中培养细胞。
在实验当天,细胞应达到80%融合。
吸出培养基并用3 ml HEPES缓冲液洗涤细胞两次。
在每个培养皿中加入3 ml HEPES缓冲液。
将细胞放回组织培养箱30分钟。缓冲液中缺乏营养素意味着在营养素刺激之前处于“饥饿”时期。
准备100μMDHA刺激缓冲液。
a。要准备20 ml的100μMDHA刺激缓冲液,请使用玻璃毛细管微量移液器将66μl的10 mg / ml DHA(30.4 mM)储备溶液添加到玻璃烧杯中。然后加入20 ml HEPES缓冲液。       


b。在刺激前立即以输出量5和恒定占空比对溶液进行超声处理3分钟,以防止DHA沉淀。如果使用其他超声仪,则将幅度设置为40%。超声处理后,溶液应澄清,且起泡最少。      


注意:使用前,请务必重新准备此DHA解决方案。


轻轻抽吸HEPES缓冲液。然后在每个对照皿中添加1ml HEPES缓冲液,在每个刺激皿中添加1ml DHA刺激缓冲液。将所有盘子放回组织培养箱中15分钟。
从每个培养皿中收集600 µl培养基,并放入新的1.5 ml微量离心管中,然后将其置于冰上。
将0.6 ofl的100 mM PMSF(请参阅食谱)以600 µM的终浓度添加到600 Ml的培养基中。  
在4°C下以850 x g 旋转微量离心管5分钟。
将500毫升的介质转移到新的试管中。
注意:现在可以将样品用于ELISA测量了。一个人还可以将样品在-80°C下保存最多两周。


根据制造商的说明,通过ELISA试剂盒测量样品中GLP-1的浓度。确保按照制造商的建议设置标准。
 


裂解培养物中的细胞,并通过Bradford分析法测量所有培养皿中的蛋白质浓度
注意:这是为了说明这些实验中每个培养皿中细胞数量的差异。


小心除去每个培养皿中任何残留的HEPES缓冲液。
在每个培养皿中加入1 ml冷PBS。
使用细胞举升机非常小心地刮下细胞。
将细胞收集到1.5 ml微量离心管中并置于冰上。
在4°C下以850 xg 旋转试管5分钟。
吸出上清液,然后加入50微升细胞裂解缓冲液,轻轻上下吸管匀浆。将试管放在冰上。
裂解细胞20分钟。每5分钟涡旋一次。
在4°C下以21,130 xg f 或15分钟旋转试管。
将上清液转移到新管中。
根据制造商的说明,使用蛋白质测定染料试剂浓缩液测量蛋白质浓度。
 


数据分析


 


从三个独立的实验中适当计算出每个样品的GLP-1浓度,每个实验重复两次。然后将该值归一化为通过Brad ford测定法测量的其相应的蛋白质浓度值(表1)。使用任何具有4参数逻辑回归的软件包进行ELISA分析。


 


表1. 调整后的GLP-1浓度


 


调整后的GLP-1


实验


 


无FA(平均GLP-1)


54.63834


 


没有FA父母


56.75393031


1个


 


DHA(平均GLP-1)


492.2011


 


没有FA父母


55.23913626


2


 


 


 


 


没有FA父母


51.92193876


3


 


 


没有FA


DHA


DHA 100家长


439.799523


1个


 


标清


2.471388


137.8699


DHA 100家长


388.2149664


2


 


扫描电镜


1.426856


79.5992


DHA 100家长


648.5889426


3


 


P 值


0.005341


 


 


笔记


 


使用健康的细胞对于进行实验至关重要。应该定期检查细胞生长状况和支原体。始终记得在氮气中填充DHA储备溶液,并在使用前新鲜准备刺激缓冲液。如果将样品在-80°C下冷冻,请勿重复冷冻和融化。


 


菜谱


 


10 mg / ml DHA储备溶液
来自制造商的600升DHA液体(1克,总计1.2毫升)
49.4毫升100%纯乙醇
分装到小玻璃瓶中,并充入氮气。储存在-20°C
HEPES缓冲液
20 mM HEPES,pH 7.9


140 毫米氯化钠


4.5 毫米氯化钾


1.2 毫米CaCl 2


1.2 毫米MgCl 2


100μMDHA刺激缓冲液
66 of 10 mg / ml DHA储备液


20 ml HEPES缓冲液


在使用前以5级恒定占空比对溶液进行超声处理3分钟


细胞裂解缓冲液
25 mM HEPES,pH 7.9


100 毫米氯化钠


1 毫米EDTA


10%甘油


1%的Triton X-100


100 mM PMSF储备溶液
174.2毫克PMSF


10 米升100%乙醇


 


致谢


 


这项工作是通过以下来源向LS-D资助的:NIH 5 K12 HD-068369-05和儿童健康临床研究咨询委员会:CCRAC 195。


该协议改编自平泽等。(2005)。


 


利益争夺


 


作者宣称没有利益冲突。


 


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引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Qi, L., Shuai, T., Da, J., Ezra, B. and Luis, S. (2020). In-vitro GLP-1 Release Assay Using STC-1 Cells. Bio-protocol 10(16): e3717. DOI: 10.21769/BioProtoc.3717.
  2. Sifuentes-Dominguez, L. F., Li, H., Llano, E., Liu, Z., Singla, A., Patel, A. S., Kathania, M., Khoury, A., Norris, N., Rios, J. J., Starokadomskyy, P., Park, J. Y., Gopal, P., Liu, Q., Tan, S., Chan, L., Ross, T., Harrison, S., Venuprasad, K., Baker, L. A., Jia, D. and Burstein, E. (2019). SCGN deficiency results in colitis susceptibility. Elife 8: e49910.
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