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Enhancement of Mucus Production in Eukaryotic Cells and Quantification of Adherent Mucus by ELISA
ELISA分析真核细胞中粘液生成增强并定量粘附的粘液   

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Cellular Microbiology
Nov 2017

Abstract

The mucosal surfaces of the gastrointestinal, respiratory, reproductive, and urinary tracts, and the surface of the eye harbor a resident microflora that lives in symbiosis with their host and forms a complex ecosystem. The protection of the vulnerable epithelium is primarily achieved by mucins that form a gel-like structure adherent to the apical cell surface. This mucus layer constitutes a physical and chemical barrier between the microbial flora and the underlying epithelium. Mucus is critical to the maintenance of a homeostatic relationship between the microbiota and its host. Subtle deviations from this dynamic interaction may result in major implications for health. The protocol in this article describes the procedures to grow low mucus-producing HT29 and high mucus-producing HT29-MTX-E12 cells, maintain cells and use them for mucus quantification by ELISA. Additionally, it is described how to assess the amount of secreted adherent mucus. This system can be used to study the protective effect of mucus, e.g., against bacterial toxins, to test the effect of different culture conditions on mucus production or to analyze diffusion of molecules through the mucus layer. Since the ELISA used in this protocol is available for different species and mucus proteins, also other cell types can be used.

Keywords: Mucus (粘液), Mucin (粘蛋白), ELISA (ELISA), Cell culture (细胞培养), HT29 (HT29)

Background

The interface of the body with the external environment is formed by mucosal surfaces. These mucosal epithelial tissues can be found for example in the gastrointestinal, respiratory, reproductive, and urinary tracts, and the surface of the eye. Due to their exposure to the external environment many microorganisms populate these tissues. Therefore, these epithelia have evolved multiple mechanisms of defense in response to their vulnerability to microbial attack. Many defensive compounds are secreted into the mucosal fluid, including mucins, antibodies, defensins, protegrins, collectins, cathelicidins, lysozyme, histatins, and nitric oxide (Kagnoff and Eckmann, 1997; Lu et al., 2002; Raj and Dentino, 2002).

To date, more than 20 genes encoding mucins have been identified in humans (Corfield, 2015). The human mucin (MUC) family comprises membrane-bound (MUC1, MUC3A/B, MUC4, MUC12, MUC13, MUC15 – 17, MUC20 and MUC21) and secreted mucins (MUC2, MUC5AC, MUC5B, MUC6 – 9, MUC19) (Moran et al., 2011; Tailford et al., 2015).

The mucus layer of the intestinal epithelial surface is mainly composed of the secreted mucin MUC2, but the membrane-bound mucins MUC1, MUC3 and MUC4 are also expressed (Kim and Ho, 2010). In addition, the intestinal mucus layer differs in terms of composition, organization and thickness along the gastrointestinal tract (Tailford et al., 2015). The secreted mucins form a gel-like structure adherent to the apical cell surface that constitutes a physical and chemical barrier between the luminal contents and the underlying epithelium (Allan, 2011). Inflammasome activity controls the secretion of mucus in goblet cells and increased secretion of mucus is observed as the microbiota becomes more diverse (Jakobsson et al., 2015). It is becoming apparent that mucus plays a crucial role in maintaining a homeostatic balance between microbiota and its host. Even small deviations from this dynamic interaction can have significant health effects, among which are colitis, colorectal cancer and susceptibility to infection (McGuckin et al., 2011; Hansson, 2012; Chen and Stappenbeck, 2014).

In this protocol, we describe the culture of in vitro models producing different amounts of mucus depending on their culture condition (static vs. semi-wet with mechanical stimulation (Navabi et al., 2013). These models are based on the little to no mucus-producing HT29 cell line, a human colon adenocarcinoma cell line, and its high mucus-producing derivative HT29-MTX-E12 (E12). Furthermore, it is described how to quantify the mucus produced in the different models by ELISA. In addition, the mucolytic compound N-acetyl-L-cysteine (NAC) is used to remove adherent mucus in order to quantify the amount of secreted adherent mucus. A schematic overview of the workflow described in this protocol is provided in Figure 1.

The method described in this protocol is suitable to study the protective effect of mucus against bacterial toxins (Reuter et al., 2018), to test the effect of different culture conditions on mucus production (Navabi et al., 2013) or to analyze diffusion of molecules through the mucus layer. Since the ELISA used in this protocol is available for different species and mucus proteins, other cell types can also be used.

Materials and Reagents

  1. 1.5 ml microcentrifuge tubes (SARSTEDT, catalog number: 72.690.001 )
  2. 15 ml centrifuge tubes (Corning, Falcon®, catalog number: 352196 )
  3. Absorbent paper
  4. 175 cm2 flask (Greiner Bio One International, CellStar®, catalog number: 660160 )
  5. Transwell® Inserts with 0.4 µm porous polyester membranes and 12 mm diameter (Corning, Transwell®, catalog number: 3460 )
  6. 12-well plate (included in Corning Transwell® package; if additional plates are required: Corning, Costar®, catalog number: 3513 )
  7. Cell lines HT29 (European Collection of Authenticated Cell Cultures (ECACC), catalog number: 91072201 ) and HT29-MTX-E12 (European Collection of Authenticated Cell Cultures (ECACC), catalog number: 12040401 ) or other cells to be tested for their mucus production
  8. Deionized water
  9. 70% ethanol
  10. Dulbecco’s modified Eagle’s medium (DMEM), high glucose, GlutaMAX (Thermo Fisher Scientific, GibcoTM, catalog number: 31966021 )
  11. Heat-inactivated fetal calf serum (FCS)
  12. 100x Penicillin/Streptomycin solution (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
  13. 100x Non-essential Amino Acids (Thermo Fisher Scientific, GibcoTM, catalog number: 11140050 )
  14. Phosphate buffered saline (PBS), pH 7.0-7.2 (Thermo Fisher Scientific, GibcoTM, catalog number: 14190144 )
  15. 0.05% Trypsin-EDTA solution (Thermo Fisher Scientific, GibcoTM, catalog number: 25300054 )
  16. ELISA kit for mucin (CLOUD-CLONE, catalog number: SEA705Hu )
    Note: In this protocol, the ELISA kit SEA705Hu was used for measurement of human mucin 2 (MUC2). Kits for different species and mucus proteins are available at Cloud-Clone Corp., e.g., SEA413Mu for measurement of mouse mucin 1 (MUC1)).
  17. N-acetyl-L-cysteine (Sigma-Aldrich, catalog number: A9165 )
  18. Cell culture medium (see Recipes)
  19. N-acetyl-L-cysteine working solution (see Recipes)

Equipment

  1. Sterile forceps
  2. Container for wash solution
  3. Multichannel Pipette (volume range: 20-200 µl)
  4. Water bath
  5. Humidified CO2 incubator (Thermo Fisher Scientific, model: HeracellTM 150i )
  6. Biological safety cabinet
  7. Hemacytometer (BRAND, Neubauer Improved, catalog number: 717805 )
  8. 37 °C incubator (e.g., CO2 incubator at 37 °C with CO2 switched off)
  9. Orbital shaker for use in CO2 incubators (Infors, model: Celltron )
  10. Ultrasonicator (Ultrasonic Homogenizer, BioLogics, model: 300VT )
  11. Microcentrifuge (Eppendorf, model: 5418 )
  12. Swing Bucket Centrifuge (Thermo Fisher Scientific, model: HeraeusTM MegafugeTM 16R )
  13. Microplate reader (Tecan Trading, model: Infinite® 200 )

Procedure

See Figure 1 for an overview of the workflow described in this protocol.


Figure 1. Schematic overview of the workflow described in this protocol. A. The procedure how to thaw, expand, and maintain HT29 and HT29-MTX-E12 (E12) cells in culture is described in Procedure A of this protocol. B. The generation and maintenance of models based on Transwell inserts, and how to initiate the different culture conditions (static vs. semi-wet interface with mechanical stimulation (SWMS)) is specified in Procedure B. C. In Procedure C the removal of adherent mucus and the preparation of models for adherent mucus quantification is described. D. The procedure of the mucus ELISA with subsequent data acquisition and analysis is explained in Procedure D and section “Data analysis” of this protocol.

  1. Growing and maintaining of HT29 and HT29-MTX-E12 (E12) cells
    Note: Before you start, heat the cell culture medium to 37 °C.
    1. Rapidly thaw a vial of frozen HT29 and HT29-MTX-E12 (E12) cells containing 1 x 106 cells by gentle agitation in a 37 °C water bath. To reduce the possibility of contamination, keep the cap of the tube out of the water. Remove the vial from the water bath as soon as the contents are thawed, and decontaminate carefully with 70% ethanol. From this point on all further steps should be carried out under strict aseptic conditions.
    2. Transfer the vial contents to a 15 ml centrifuge tube containing 9 ml warm cell culture medium and centrifuge at 300 x g for 5 min at room temperature with maximum acceleration/deceleration settings. Discard supernatant. Resuspend cells in 1 ml pre-warmed medium and transfer the cell suspension to a 175 cm2 flask containing 25 ml pre-warmed medium. Place the flask in a humidified CO2 forced-air incubator at 37 °C.
    3. Twenty four hours after seeding, check the cells under a microscope for attachment, if attached to flask surface, slowly aspirate media to remove dead cells and add 25 ml fresh pre-warmed medium. Change the medium every 2-3 days.
    4. When the cells have reached 80% confluency, they are ready for passaging. Cells need to be passaged for a minimum of 3 cycles before using them for the experiment.
      Note: This is necessary to give the cells time to recover from thawing and may vary from cell type to cell type. The HT29 and E12 cells applied in this protocol were used until passage 20.
    5. For passaging, remove media and wash monolayer with 10 ml PBS. Add 5 ml of pre-warmed (37 °C) trypsin-EDTA solution and incubate at 37 °C for 3-10 min. Check the detachment of cells regularly under the microscope. When cells start to detach, add 5 ml warm media to inactivate trypsin and thoroughly pipette several times to get a uniform cell suspension (mucus-producing cells tend to form cellular lumps).
      Note: The incubation time with trypsin-EDTA solution and subsequent detachment of cells varies from cell type to cell type and detachment of cells should be controlled under the microscope regularly to avoid excessively long incubation time. The detachment process can be quickened by gently knocking with the hand against the cell culture flask.
    6. Transfer the cell suspension into a 15 ml centrifuge tube and centrifuge at 300 x g for 5 min at room temperature. Discard supernatant and resuspend cells in 5 ml pre-warmed medium. Count cells using a hemacytometer and transfer 7 x 105 cells into a new 175 cm2 flask with 25 ml fresh pre-warmed medium. The remaining cells can be used for subsequent experiments.

  2. Setup and culture of cell culture models
    1. Unpack the Transwell® inserts and place them in the cavities of a 12-well plate. Subsequently, harvest and count HT29 and E12 cells as described in Steps A5 and A6. Adjust the cell concentration of the obtained cell suspension to a working concentration of 2.73 x 105 cells per ml.
    2. To set up the models, add 275 µl of the cell suspension containing 7.5 x 104 HT29 or E12 cells to the apical side of the membrane and 1 ml medium to the basal compartment. Incubate the plates in the cell culture incubator overnight.
    3. On the next day, the medium is changed and the culture condition semi-wet interface with mechanical stimulation (SWMS) is initiated to enhance mucus production.
      1. To generate the static culture condition, replace the medium in the apical compartment with 275 µl and the basal compartment with 1 ml pre-warmed (37 °C) medium as described in Step B4. Incubate the cells in a cell culture incubator for 15 days and maintain the media volumes during the medium change.
      2. To generate culture condition semi-wet interface with mechanical stimulation (SWMS), replace the medium in the apical compartment with 75 µl and the basal compartment with 850 µl pre-warmed (37 °C) medium as described in Step B4. Incubate the cells on an orbital shaker with an agitation of 65 rpm in a cell culture incubator for 15 days and maintain the media volumes during the medium change.
    4. A medium exchange should be done every 1-2 days as secreted mucus will acidify the medium (the medium turns yellow). To change the medium, aspirate the medium in the basal compartment first. Then, use sterile forceps to lift out the Transwell® inserts and very carefully aspirate the apical medium without disturbing the cells or the adherent mucus layer. Subsequently, fill very carefully 275 µl or 75 µl of fresh pre-warmed medium into the apical compartment and put the inserts back into their cavities. Finally, fill 1 ml or 850 µl into the lower compartment and hold the plate at a slight angle to avoid air bubbles underneath the membrane. The medium in the inner and outer compartments should have the same level.

  3. Preparation of models for mucus quantification by ELISA
    1. After 15 days, the models are ready and can be used for experiments. If quantification of secreted adherent mucus is required a subset of models should be treated as described in Step C2, otherwise continue with Step C3.
      Notes:
      1. Perceivable ideas for experiments could be to test the colonization efficacy of bacteria in presence and absence of an adherent mucus layer, or to test the diffusion of particles through the cell layer in presence and absence of an adherent mucus layer.
      2. If other cell types are used, the optimal culture time for maximal mucus production can be determined by performing the mucus ELISA at different time points. In addition, histological sections can be prepared and stained with PAS/Alcian blue to stain mucus and to obtain a qualitative overview of the mucus localization in the cell layer.
    2. The mucolytic agent N-acetyl-L-cysteine (NAC) is used to chemically remove adherent mucus. NAC has a low order of toxicity and reduces the viscosity of mucus by irreversible reduction of disulfide bonds in mucoproteins.
      1. For this purpose, remove the medium as described in Step B4.
      2. Add 500 µl pre-warmed (37 °C) NAC working solution to the apical compartment and 1.5 ml pre-warmed (37 °C) medium to the basal compartment in order to balance the medium level in both compartments. Subsequently, place the prepared models on an orbital shaker for 1 h and 65 rpm in the cell culture incubator.
      3. Wash both compartments once with PBS and continue with Step C3.
    3. To detach cells, carefully aspirate the medium as described in Step B4 and carefully wash both compartments once with PBS by adding 500 µl to the apical and 1.5 ml to the basal compartment.
    4. Remove PBS as described in Step B4 and add pre-warmed (37 °C) trypsin-EDTA solution (500 µl apical, 1.5 ml basal) to each model. Incubate at 37 °C for 10-15 min in the cell culture incubator.
    5. Detach cells by thoroughly pipetting several times to get a uniform cell suspension and transfer the solution into a 1.5 ml tube containing 500 µl medium. Subsequently, count cells in a hemacytometer.
      Note: When the cells begin to detach, the fluid becomes cloudy and small “holes” become visible on the membrane.
    6. An equal number of cells should be analyzed by ELISA. Therefore, transfer 4 x 106 cells per approach into a new 1.5 ml tube and centrifuge at 2,000 x g for 5 min at 4 °C. Resuspend pellet in 500 µl ice-cold PBS. Subsequently, subject the cell suspension to ultrasonication for 4 times 10 sec at 20 kHz on ice (avoid foaming).
      Note: The number of cells to be analyzed can be varied and should be roughly estimated in advance on the basis of the mucus production of the cells to achieve optimal results in the ELISA.
    7. Centrifuge at 1,500 x g for 10 min at 4 °C to remove cellular debris. The supernatant is required for ELISA. Keep samples on ice until use.

  4. ELISA and data acquisition
    1. In the following step, the assay is performed according to the manufacturer's instructions. Reconstitute the standard included in the kit and prepare a dilution series with 7 points. Reconstitute all other components included in the kit according to the manufacturer's instructions.
    2. Perform the assay in duplicate readings for each standard, blank (Standard Diluent, included in the kit), and samples. Load the pre-coated ELISA-plate with 100 µl of standard, blank, and samples and incubate for 2 h at 37 °C. Remove the liquid of each well, don’t wash. Add primary antibody and incubate for 1 h at 37 °C. Wash each well three times with wash solution and incubate with HRP-conjugated secondary antibody for 30 min at 37 °C. Wash each well again five times and subsequently incubate with 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate solution for 15-25 min at 37 °C in the dark. Add sulphuric acid to stop the reaction and measure absorption immediately at 450 nm with a Microplate Reader.
      Note: The liquid will turn blue by the addition of TMB substrate. The incubation time with TMB substrate is critical and may vary. It is therefore strongly recommended to check the course of the reaction regularly. The strongest blue coloration should be observed at the highest standard. In this protocol, approximately 20 min is sufficient. The liquid will turn yellow by the addition of Stop solution. Mix the liquid until uniform by tapping the side of the plate. In some cases, dilution is required before measurement to obtain optimal measurement results.

Data analysis

  1. A typical measurement is shown in Figure 2A. First, average the duplicate readings for each standard, blank, and samples and subtract the average blank optical density. Next, draw a standard curve by plotting the mean optical density (OD) for each standard on the linear y-axis and mucus concentration on the linear x-axis. Create a linear regression line and use the formula of the regression line to calculate the mucus concentrations from the averaged measured OD values of the samples (Figures 2B and 2C). If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.
  2. To calculate the amount of secreted mucus, the mucus concentration of the NAC-treated samples must be subtracted from the mucus concentration of the untreated samples. The difference represents the amount of secreted mucus (Figure 2C).


    Figure 2. Exemplary data analysis of measurement with MUC2 ELISA in HT29 and HT29-MTX-E12 (E12) cells under different culture conditions. A. A typical absorption measurement with a Microplate Reader at 450 nm is shown after averaging the duplicate reads of blank, standard, and samples. B. The incubation time with TMB substrate was 20 min. Using the measured OD450 values for the standard, a standard curve can be created after subtraction of the averaged blank value. By inserting a linear regression line, the mucus concentration of the samples can be calculated using the regression line equation. C. The calculated mucus concentration for the samples is shown in and after subtraction of the NAC-treated samples the amount of secreted mucus can be determined.

Recipes

  1. Cell culture medium (500 ml)
    440 ml DMEM, high glucose, GlutaMAX
    50 ml FCS
    5 ml Penicillin/Streptomycin
    5 ml non-essential amino acids
  2. N-acetyl-L-cysteine working solution (60 mM)
    10 ml DMEM, high glucose, GlutaMAX
    97.9 mg N-acetyl-L-cysteine

Acknowledgments

This protocol is adapted from Reuter et al. (2018). The work was partly supported by Ardeypharm GmbH. The authors do not have any potential conflicts of interest to declare.

References

  1. Allan, A. (2011). Gastrointestinal mucus. Compr Physiol 359-382.
  2. Chen, G.Y. and Stappenbeck, T.S. (2014). Mucus, it is not just a static barrier. Sci Signal 7, pe11.
  3. Corfield, A. P. (2015). Mucins: a biologically relevant glycan barrier in mucosal protection. Biochim Biophys Acta 1850(1): 236-252.
  4. Hansson, G. C. (2012). Role of mucus layers in gut infection and inflammation. Curr Opin Microbiol 15(1): 57-62.
  5. Jakobsson, H. E., Rodriguez-Pineiro, A. M., Schutte, A., Ermund, A., Boysen, P., Bemark, M., Sommer, F., Backhed, F., Hansson, G. C. and Johansson, M. E. (2015). The composition of the gut microbiota shapes the colon mucus barrier. EMBO Rep 16(2): 164-177.
  6. Kagnoff, M.F. and Eckmann, L. (1997). Epithelial cells as sensors for microbial infection. J Clin Invest 100: 6-10.
  7. Kim, Y. S. and Ho, S. B. (2010). Intestinal goblet cells and mucins in health and disease: recent insights and progress. Curr Gastroenterol Rep 12(5): 319-330.
  8. Lu, J., Teh, C., Kishore, U. and Reid, K. B. (2002). Collectins and ficolins: sugar pattern recognition molecules of the mammalian innate immune system. Biochim Biophys Acta 1572(2-3): 387-400.
  9. McGuckin, M. A., Linden, S. K., Sutton, P. and Florin, T. H. (2011). Mucin dynamics and enteric pathogens. Nat Rev Microbiol 9(4): 265-278.
  10. Moran, A. P., Gupta, A. and Joshi, L. (2011). Sweet-talk: role of host glycosylation in bacterial pathogenesis of the gastrointestinal tract. Gut 60(10): 1412-1425.
  11. Navabi, N., McGuckin, M.A. and Linden, S.K. (2013). Gastrointestinal cell lines form polarized epithelia with an adherent mucus layer when cultured in semi-wet interfaces with mechanical stimulation. PLoS One 8: e68761.
  12. Raj, P. A. and Dentino, A. R. (2002). Current status of defensins and their role in innate and adaptive immunity. FEMS Microbiol Lett 206(1): 9-18.
  13. Reuter, C., Alzheimer, M., Walles, H. and Oelschlaeger, T.A. (2018). An adherent mucus layer attenuates the genotoxic effect of colibactin. Cell Microbiol 20.
  14. Tailford, L. E., Crost, E. H., Kavanaugh, D. and Juge, N. (2015). Mucin glycan foraging in the human gut microbiome. Front Genet 6: 81.

简介

胃肠道,呼吸道,生殖道和泌尿道的粘膜表面以及眼睛表面都有一个居住的微生物群落,它们与宿主共生并形成一个复杂的生态系统。脆弱的上皮细胞的保护主要通过形成附着于顶端细胞表面的凝胶样结构的粘蛋白实现。该粘液层构成了微生物菌群和下层上皮之间的物理和化学屏障。粘液对维持微生物群与宿主之间的稳态关系至关重要。与这种动态互动的细微差异可能会对健康产生重大影响。本文中的方案描述了生长低粘液产生HT29和高粘液产生HT29-MTX-E12细胞的程序,维持细胞并通过ELISA将其用于粘液定量。此外,还介绍了如何评估分泌黏液的数量。该系统可用于研究粘液对抗细菌毒素的保护作用,例如测试不同培养条件对粘液产生的影响或分析分子通过粘液层的扩散。由于本方案中使用的ELISA可用于不同的物种和粘液蛋白,因此也可以使用其他细胞类型。

【背景】身体与外部环境的界面由粘膜表面形成。这些粘膜上皮组织可以在例如胃肠道,呼吸道,生殖道和尿道以及眼睛表面发现。由于它们暴露于外部环境中,许多微生物会聚集在这些组织中。因此,这些上皮细胞已经进化出多种防御机制来回应其易受微生物攻击的影响。许多防御性化合物被分泌到粘膜液中,包括粘蛋白,抗体,防御素,protegrin,聚集蛋白,cathelicidins,溶菌酶,组蛋白和一氧化氮(Kagnoff和Eckmann,1997,Lu等人,2002 ,Raj和Dentino,2002年)。

迄今为止,人类已经发现了超过20种编码粘蛋白的基因(Corfield,2015)。人粘蛋白(MUC)家族包含膜结合的(MUC1,MUC3A / B,MUC4,MUC12,MUC13,MUC15-17,MUC20和MUC21)和分泌的粘蛋白(MUC2,MUC5AC,MUC5B,MUC6-9,MUC19) ,2011,Tailford 等,2015)。

肠上皮表面的粘液层主要由分泌的粘蛋白MUC2组成,但是膜结合的粘蛋白MUC1,MUC3和MUC4也表达(Kim和Ho,2010)。另外,肠粘液层在胃肠道的组成,组织和厚度方面有所不同(Tailford等人,2015)。分泌的粘蛋白形成粘附于顶端细胞表面的凝胶样结构,其构成腔内容物和下层上皮之间的物理和化学屏障(Allan,2011)。炎性体活动控制杯状细胞中的粘液分泌,并且随着微生物群变得更加多样化,观察到粘液分泌增加(Jakobsson等人,2015)。越来越明显的是,粘液在维持微生物群与宿主之间的稳态平衡方面发挥着至关重要的作用。即使与这种动态相互作用的微小偏差也可能具有显着的健康影响,其中包括结肠炎,结肠直肠癌和感染易感性(McGuckin等,2011,Hansson,2012,Chen and Stappenbeck,2014)。

在这个协议中,我们描述了根据培养条件产生不同数量粘液的体外培养模型(静态与半湿态与机械刺激(Navabi等人 “,2013),这些模型是基于少量或不产生粘液的HT29细胞系,一种人类结肠腺癌细胞系及其高产粘液衍生物HT29-MTX-E12(E12)。通过ELISA定量不同模型中产生的粘液,此外,粘液溶解化合物N-乙酰-L-半胱氨酸(NAC)用于去除粘附粘液以定量分泌的粘附粘液的量。图1中提供了此协议中描述的工作流程。

本方案中描述的方法适用于研究粘液对细菌毒素的保护作用(Reuter等人,2018年),以测试不同培养条件对粘液产生的影响(Navabi等人, 2013)或分析通过粘液层的分子扩散。由于本方案中使用的ELISA可用于不同的物种和粘液蛋白,也可以使用其他细胞类型。

关键字:粘液, 粘蛋白, ELISA, 细胞培养, HT29

材料和试剂


  1. 1.5 ml微量离心管(SARSTEDT,目录号:72.690.001)
  2. 15ml离心管(Corning,Falcon ,目录号:352196)
  3. 吸水纸
  4. 175cm 2烧瓶(Greiner Bio One International,CellStar,目录号:660160)。
  5. 带有0.4μm多孔聚酯膜和12mm直径的Transwell插件(Corning,Transwell ,目录号:3460)
  6. 12孔板(包含在康宁Transwell 包装中;如果需要附加板:康宁,Costar ®,产品目录号:3513)
  7. 细胞系HT29(European Collection of Authenticated Cell Cultures(ECACC),目录号:91072201)和HT29-MTX-E12(European Collection of Authenticated Cell Cultures(ECACC),目录号:12040401)或其他待测试其粘液生产
  8. 去离子水
  9. 70%乙醇
  10. Dulbecco改良的Eagle培养基(DMEM),高葡萄糖,GlutaMAX(Thermo Fisher Scientific,Gibco TM,产品目录号:31966021)
  11. 热灭活的胎牛血清(FCS)
  12. 100x青霉素/链霉素溶液(Thermo Fisher Scientific,Gibco TM,目录号:15140122)
  13. 100x非必需氨基酸(Thermo Fisher Scientific,Gibco TM,产品目录号:11140050)
  14. 磷酸盐缓冲盐水(PBS),pH 7.0-7.2(Thermo Fisher Scientific,Gibco TM,目录号:14190144)
  15. 0.05%胰蛋白酶-EDTA溶液(Thermo Fisher Scientific,Gibco TM,目录号:25300054)
  16. 用于粘蛋白的ELISA试剂盒(CLOUD-CLONE,目录号:SEA705Hu)
    注意:在该方案中,ELISA试剂盒SEA705Hu用于测量人粘蛋白2(MUC2)。用于不同物种和粘液蛋白的试剂盒可在Cloud-Clone Corp.获得,例如SEA413Mu用于测量小鼠粘蛋白1(MUC1))。
  17. N-乙酰-L-半胱氨酸(Sigma-Aldrich,目录号:A9165)
  18. 细胞培养基(见食谱)
  19. N-乙酰-L-半胱氨酸工作溶液(见食谱)

设备

  1. 无菌钳
  2. 洗涤液容器
  3. 多道移液器(体积范围:20-200μl)
  4. 水浴
  5. 加湿的CO 2培养箱(Thermo Fisher Scientific,型号:Heracell TM 150i)
  6. 生物安全柜
  7. 血细胞计数器(品牌,Neubauer改进,目录号:717805)
  8. 37℃培养箱(例如,CO 2培养箱,37℃,CO 2关闭)。
  9. 用于CO 2孵化器的轨道振荡器(Infors,型号:Celltron)
  10. 超声波发生器(Ultrasonic Homogenizer,BioLogics,型号:300VT)
  11. 微量离心机(Eppendorf,型号:5418)
  12. Swing桶式离心机(Thermo Fisher Scientific,型号:Heraeus TM Megafuge TM 16R)
  13. 酶标仪(Tecan Trading,型号:Infinite <200>)

程序


请参见图1,了解此协议中描述的工作流程概述

图1.本协议中描述的工作流程示意图概述A.如何在培养物中解冻,扩增和维持HT29和HT29-MTX-E12(E12)细胞的程序在程序A中描述的协议。 B.基于Transwell插入物的模型的生成和维护以及如何启动不同的培养条件(静态与半湿润界面与机械刺激(SWMS))在程序BC中规定。在程序C中,去除粘附的粘液和描述了用于粘附性粘液定量的模型的制备。 D.在随后的数据采集和分析中进行的粘液ELISA的程序在本程序的程序D和“数据分析”部分中进行了解释。

  1. 生长和维持HT29和HT29-MTX-E12(E12)细胞
    注意:开始之前,将细胞培养基加热至37°C。
    1. 通过在37℃水浴中温和搅拌,快速解冻一小瓶含有1×10 6个细胞的冷冻HT29和HT29-MTX-E12(E12)细胞。为了减少污染的可能性,请将管的盖子从水中取出。一旦内容物解冻,立即从水浴中取出小瓶,并用70%乙醇仔细去污。从这一点开始,所有进一步的步骤都应该在严格的无菌条件下进行。
    2. 将小瓶内容物转移至含有9ml温热细胞培养基的15ml离心管中,并在室温下以300Xg g离心5分钟,同时进行最大加速/减速设定。弃上清。将细胞重悬于1ml预热培养基中,并将细胞悬浮液转移至含有25ml预热培养基的175cm 2烧瓶中。
      将烧瓶置于37℃的湿润CO 2强制空气培养箱中。
    3. 接种24小时后,在显微镜下检查细胞是否附着,如果附着于烧瓶表面,则缓慢吸出培养基以除去死细胞并加入25ml新鲜预热培养基。每2-3天更换一次媒体。
    4. 当细胞达到80%融合时,它们可以进行传代。
      在将细胞用于实验之前,细胞需要传代至少3个周期 注意:这是使细胞有时间从融化中恢复的必要条件,并且可能因细胞类型而异。使用本协议中使用的HT29和E12细胞直到第20代。
    5. 为了传代,移出培养基并用10ml PBS洗涤单层。加入5ml预热(37℃)胰蛋白酶-EDTA溶液并在37℃下孵育3-10分钟。在显微镜下定期检查细胞脱落。当细胞开始分离时,加入5 ml温培养基以灭活胰蛋白酶并彻底吸移几次以获得均匀的细胞悬液(产生粘液的细胞倾向于形成细胞团块)。
      注意:胰蛋白酶-EDTA溶液的孵育时间和随后细胞的分离随着细胞类型的不同而不同,细胞的分离应定期在显微镜下控制以避免过长的孵育时间。

      通过用手轻轻敲打细胞培养瓶,可以加快分离过程。
    6. 将细胞悬浮液转移到15ml离心管中并在室温下300×g离心5分钟。弃去上清液并重悬于5ml预热培养基中的细胞。使用血细胞计数器计数细胞并将7×10 5个细胞转移到具有25ml新鲜预热培养基的新的175cm 2烧瓶中。剩下的细胞可以用于后续的实验。

  2. 细胞培养模型的设置和培养
    1. 打开Transwell ®插件的包装并将它们放入12孔板的空腔中。随后,如步骤A5和A6中所述收获并计数HT29和E12细胞。将获得的细胞悬浮液的细胞浓度调节至每毫升2.73×10 5个细胞的工作浓度。
    2. 为了建立模型,将275μl含有7.5×10 4 HT29或E12细胞的细胞悬液加入到膜的顶侧,并将1ml培养基加入到基底隔室中。
      培养细胞培养箱中的培养板过夜
    3. 第二天,改变培养基并启动具有机械刺激(SWMS)的培养条件半湿界面以增强粘液产生。
      1. 为了产生静态培养条件,如步骤B4所述,用275μl和顶部隔室用1ml预热(37℃)培养基代替顶部隔室中的培养基。
        在细胞培养培养箱中培养细胞15天,并在培养基更换期间保持培养基体积。
      2. 为了产生具有机械刺激(SWMS)的培养条件半湿界面,如步骤B4所述,用75μl和顶部隔室中的850μl预热(37℃)培养基代替顶部隔室中的培养基。在细胞培养箱中以65rpm的速度将细胞孵育在轨道摇床上15天,并在培养基更换期间保持培养基体积。
    4. 每隔1-2天应进行一次培养基交换,因为分泌的粘液会使培养基酸化(培养基变黄)。要更换培养基,请首先吸入基底隔室中的培养基。然后,使用无菌镊子将Transwell™插入物取出并非常小心地吸出顶端介质,而不会干扰细胞或贴壁粘液层。随后,非常小心地将275μl或75μl新鲜的预热培养基填充到顶端隔室中,并将插入物放回其腔体中。最后,将1 ml或850μl填充到下部隔室中,并将其保持一定角度以避免隔膜下方出现气泡。内部和外部隔室中的介质应具有相同的高度。

  3. 通过ELISA制备粘液定量模型
    1. 15天后,模型已准备就绪,可用于实验。如果需要定量分泌粘液粘液,则应如步骤C2所述处理模型子集,否则继续步骤C3。
      备注:
      1. 对于实验而言,可感知的想法可以是在存在和不存在粘附性粘液层的情况下测试细菌的定殖效力,或测试存在和不存在粘附性粘液层时颗粒通过细胞层的扩散。
      2. 如果使用其他细胞类型,可通过在不同时间点进行粘液ELISA来确定最大粘液产生的最佳培养时间。此外,可以制备组织切片并用PAS /阿尔新蓝染色以染色粘液并获得细胞层中粘液定位的定性概述。
    2. 粘液溶解剂N-乙酰-L-半胱氨酸(NAC)用于化学去除粘附的粘液。 NAC具有低毒性,并通过粘蛋白中二硫键的不可逆还原降低了粘液的粘度。
      1. 为此,请按照步骤B4所述移除介质。
      2. 将500μl预热(37℃)NAC工作溶液加入到顶室中,并将1.5ml预热(37℃)的培养基加入基底室以平衡两个室中的中等水平。随后,将准备好的模型置于轨道振动器上1小时,然后在细胞培养箱中转速为65转/分钟。
      3. 用PBS清洗两个隔室一次并继续步骤C3。
    3. 为了分离细胞,如步骤B4中所述小心地吸出培养基并小心地用PBS将两个隔室洗涤一次,向顶端加入500μl,并向基底隔室加入1.5ml。
    4. 如步骤B4所述去除PBS,并向每个模型添加预热(37℃)胰蛋白酶-EDTA溶液(500μl顶端,1.5ml基底)。在细胞培养箱中37°C孵育10-15分钟。
    5. 通过彻底吸取细胞数次以分离细胞以获得均匀的细胞悬液并将溶液转移至含有500μl培养基的1.5ml管中。随后,计数血细胞计数器中的细胞。
      注意:当细胞开始分离时,流体变得浑浊,膜上可见小的“孔”。
    6. 应通过ELISA分析相同数量的细胞。因此,将每种方法的4×10 6个细胞转移到新的1.5ml管中,并在4℃下以2000×g离心5分钟。在500μl冰冷的PBS中重悬沉淀。随后,使细胞悬液在冰上以20kHz超声处理4次10秒(避免起泡)。
      注意:要分析的细胞数量可以变化,并且应该基于细胞的粘液产生预先粗略估计,以在ELISA中实现最佳结果。
    7. 在4℃下以1500 xg g离心10分钟以除去细胞碎片。 ELISA需要上清液。使用前将样品保存在冰上。

  4. ELISA和数据采集
    1. 在以下步骤中,根据制造商的说明进行测定。重新构建试剂盒中包含的标准,并用7分制备稀释系列。
      根据制造商的说明重新构建套件中包含的所有其他组件
    2. 对每个标准进行重复读数的实验,空白(标准稀释剂,包括在试剂盒中)和样品。将预先包被的ELISA板加入100μl标准品,空白和样品,并在37℃孵育2小时。除去每口井的液体,不要清洗。加入一抗并在37℃下孵育1小时。用洗涤溶液将每个孔清洗三次,并在37°C下与HRP缀合的二抗孵育30分钟。每次再次洗涤5次,随后在37℃在黑暗中与3,3',5,5'-四甲基联苯胺(TMB)底物溶液一起温育15-25分钟。加入硫酸终止反应,并用微孔板读数仪立即在450 nm处测量吸收。
      注意:添加TMB底物会使液体变蓝。 TMB底物的孵育时间很关键,可能会有所不同。因此强烈建议定期检查反应过程。最高标准应该观察到最强的蓝色着色。在这个协议中,大约20分钟就足够了。加入Stop溶液后液体会变黄。通过敲击板的侧面将液体混合至均匀。在某些情况下,测量前需要稀释以获得最佳测量结果。

数据分析

  1. 典型的测量结果如图2A所示。首先,平均每个标准品,空白和样品的重复读数,并减去平均空白光密度。接下来,绘制标准曲线,绘制线性y轴上每个标准的平均光密度(OD)和线性x轴上的粘液浓度。创建一个线性回归线,并使用回归线公式计算样本的平均测量OD值的粘液浓度(图2B和2C)。如果样品被稀释,从标准曲线读取的浓度必须乘以稀释倍数。
  2. 为了计算分泌粘液的量,必须从未处理的样品的粘液浓度中减去NAC处理的样品的粘液浓度。差异代表分泌粘液的量(图2C)。


    图2.在不同培养条件下HT29和HT29-MTX-E12(E12)细胞中使用MUC2ELISA进行的测量的示例性数据分析A.显示在450nm下使用微孔板读数器的典型吸收测量值平均重复读取空白,标准和样本后。 B.与TMB底物的孵育时间为20分钟。使用标准的OD450测量值,可以在减去平均空白值后创建标准曲线。通过插入线性回归线,可以使用回归线方程计算样品的粘液浓度。 C.在减去NAC处理的样品之后和之后显示样品的计算的粘液浓度,可以确定分泌的粘液的量。

食谱

  1. 细胞培养基(500毫升)
    440毫升DMEM,高糖,GlutaMAX
    50毫升FCS
    5毫升青霉素/链霉素
    5毫升非必需氨基酸
  2. N-乙酰-L-半胱氨酸工作溶液(60 mM)
    10毫升DMEM,高糖,GlutaMAX
    97.9毫克N-乙酰-L-半胱氨酸

致谢

该协议改编自Reuter 等人(2018)。这项工作得到了Ardeypharm GmbH的部分支持。作者没有任何潜在的利益冲突声明。

参考

  1. Allan,A。(2011)。 消化道粘液 Compr Physiol ,359-
  2. 陈,G.Y.和Stappenbeck,T.S. (2014)。 粘液,它不仅仅是一个静止的屏障。科学信号 7,pe11。
  3. Corfield,A.P。(2015)。 粘蛋白:粘膜保护中与生物相关的聚糖屏障。 Biochim Biophys Acta 1850(1):236-252。
  4. Hansson,G.C。(2012)。 黏液层在肠道感染和炎症中的作用 Curr Opin Microbiol < 15(1):57-62。
  5. Jakobsson,HE,Rodriguez-Pineiro,AM,Schutte,A.,Ermund,A.,Boysen,P.,Bemark,M.,Sommer,F.,Backhed,F.,Hansson,GC和Johansson,ME(2015) 。 肠道微生物群的组成构成了结肠黏液屏障。 EMBO Rep 16(2):164-177。
  6. Kagnoff,M.F.和Eckmann,L。(1997)。 上皮细胞作为微生物感染的传感器。
  7. Kim,Y.S.和Ho,S.B。(2010)。 健康和疾病中的肠杯状细胞和粘蛋白:最近的见解和进展 Curr Gastroenterol Rep 12(5):319-330。
  8. Lu,J.,Teh,C.,Kishore,U。和Reid,K.B.(2002)。 Collectins和ficolins:哺乳动物先天免疫系统的糖模式识别分子 Biochim Biophys Acta 1572(2-3):387-400。
  9. McGuckin,M.A.,Linden,S.K。,Sutton,P.and Florin,T.H。(2011)。 粘蛋白动态和肠道病原体 Nat Rev Microbiol 9 (4):265-278。
  10. Moran,A. P.,Gupta,A。和Joshi,L。(2011)。 甜言蜜语:宿主糖基化在胃肠道细菌发病机制中的作用 Gut 60(10):1412-1425。
  11. Navabi,N.,McGuckin,M.A.和Linden,S.K. (2013年)。 胃肠道细胞系形成极化上皮细胞时,在半培养时粘附粘液层湿润接口与机械刺激。 PLoS One 8:e68761。
  12. Raj,P.A。和Dentino,A.R。(2002)。 防御素的现状及其在先天性和适应性免疫中的作用 FEMS Microbiol Lett 206(1):9-18。
  13. Reuter,C.,Alzheimer,M.,Walles,H.和Oelschlaeger,T.A. (2018)。 粘附粘液层可减弱colibactin的遗传毒性作用。 Cell Microbiol < 20。
  14. Tailford,L.E.,Crost,E.H。,Kavanaugh,D。和Juge,N。(2015)。 人类肠道微生物菌群中的粘蛋白聚糖觅食 前台遗传 6:81。
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Reuter, C. and Oelschlaeger, T. A. (2018). Enhancement of Mucus Production in Eukaryotic Cells and Quantification of Adherent Mucus by ELISA. Bio-protocol 8(12): e2879. DOI: 10.21769/BioProtoc.2879.
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