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Adhesion and Invasion Assay Procedure Using Caco-2 Cells for Listeria monocytogenes
单核细胞增生李斯特菌对Caco-2细胞的粘附和侵袭试验程序

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Abstract

Listeria monocytogenes is an important Gram-positive foodborne pathogen that is a particular problem in ready-to-eat food. It has an ability to survive in harsh conditions like refrigeration temperatures and high salt concentrations and is known to cross intestinal, placental and blood-brain barriers. Several cancerous cell lines like cervical, liver, dendritic, intestinal and macrophages have been used to study in vitro propagation and survival of listeria in human cells. Human intestinal epithelial cells have been used to study how listeria crosses the intestinal barrier and cause infection. The protocol in this articles describes the procedures to grow Caco-2 cells, maintain cells and use them for adhesion and invasion assays. During adhesion assay the cells are incubated with listeria for 30 min but in invasion assay the cell growth is arrested at several time points after infection to monitor the growth and survival rate of listeria in cells.

Keywords: Adhesion assay(粘附试验), Invasion assay(侵袭试验), Listeria(李斯特菌), Caco-2 cells(Caco-2细胞)

Background

Listeria monocytogenes being a facultative intracellular bacterium can enter, survive and multiply in both phagocytic and non-phagocytic cells and this property of the bacterium has been extensively studied and understood. Being a food borne pathogen it enters the blood stream in humans via intestines by crossing intestinal barriers. Thus human intestinal cells are used as an in vitro medium to study adhesion and intracellular survival of Listeria monocytogenes. Human colon adenocarcinoma cells, Caco-2 cells have been used extensively as a model for intestinal barrier (Angelis and Turco, 2011).The protocol described in this article explains the procedure to grow and maintain Caco-2 cells and then infect them to study adhesion and invasion properties of listerial species. This protocol can also be used with minor changes for cells like HT-29 (human colorectal adenocarcinoma cells) and Tc7 cells (a subclone of the Caco-2 cell line have) which have also been used to study Listeria. These assays are generally used to compare the pathogenicity of listerial mutants with wide type(WT) strains (Reddy and Lawrence, 2014). Adhesion assay is straight forward where in the bacteria are incubated with Caco-2 cells for 30 min and the bacterial counts for mutants and WT strains are compared to observe any alteration in adhesion properties as a result of mutation. Bacterial counts obtained at different time points during invasion assay give more information about the survival of listeria in the human cells and the comparison of these counts between mutants and WT strains gives information about the changes in the adaptation of listeria after mutation in human cells. This protocol has been successfully used previously to study the adhesion and invasion properties of listerial strains (Jaradat and Bhunia, 2003; Lecuit, 2005; Sambuy et al., 2005; Reddy et al., 2016).

Materials and Reagents

  1. MF-Millipore filters (EMD Millipre, catalog number: SCGPU05RE )
  2. Eppendorf tubes (Eppendorf, catalog number: 022363204 )
  3. Cell culture flasks (Corning, catalog number: 3275 )
  4. Cell culture plates (12-well cell culture receiver plate, sterile) (EMD Millipre, catalog number: PIMWS1250 )
  5. Pipette tip  
  6. 15 ml culture tube
  7. Cell scrapers (Corning, catalog number: 3010 )
  8. ZapCap bottle-top filters, pore size 0.2 µm (Maine Manufacturing, catalog number: 10443421 )
  9. Serological pipettes (1, 5, 10, 25, 50 ml) (Corning, catalog numbers: 4010 , 4050 , 4100 , 4250 , 4501 )
  10. Culture tubes (Sigma-Aldrich, catalog number: T1661 )
  11. Listeria monocytogenes strains F2365 (wild type strain), and F2365∆2117(mutant strain) (Reddy et al., 2016)
  12. Caco-2 cell line (ATCC, catalog number: HTB-37 )
  13. 70% ethanol (Fisher Scientific, catalog number: BP82014 )
  14. Trypsin-EDTA solution (Sigma-Aldrich, catalog number: T3924 )
  15. Brain Heart Infusion (BHI) agar (Sigma-Aldrich, catalog number: 70138 )
  16. BHI broth (Sigma-Aldrich, catalog number: 53286 )
  17. Gentamicin (Sigma-Aldrich, catalog number: G1397 )
  18. Eagle’s minimum Essential medium (EMEM) (ATCC, catalog number: 30-2003 )
  19. Fetal bovine serum, certified, heat inactivated (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10082147 )
  20. Penicillin-streptomycin (10,000 U/ml) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
  21. Dulbecco’s phosphate buffered saline (Sigma-Aldrich, catalog number: D8537 )
  22. Triton X-100 (Sigma-Aldrich, catalog number: 234729 )
    Note: This product has been discontinued.
  23. Phosphate-buffered saline, 10x (PBS) (Sigma-Aldrich, catalog number: P5493 )
  24. cEMEM (see Recipes)
  25. 0.1% Triton-X 100 (see Recipes)
  26. 1x PBS (see Recipes)

Equipment

  1. Water bath, 37 °C (Thermo Fisher Scientific)
  2. CO2 forced-air incubator (Thermo Fisher Scientific, Thermo ScientificTM, model: FormaTM Steri-CultTM CO2 Incubators , catalog number: 3307TS)
  3. Biological safety cabinet (Thermo Fisher Scientific, Thermo ScientificTM, model: 1300 Series Class II )
  4. Inverted microscope(Nikon Instruments, model: Eclipse Ti-S )
  5. Pipette (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 4642070 )
  6. Pipettor (Daigger Scientific, model: Portable Pipet-Aid XP Pipette Controller )
  7. Hemacytometer (Fisher Scientific, catalog number: S17040 )
  8. 37 °C incubator (Thermo Fisher Scientific)
  9. 37°C shaker (Thermo Fisher Scientific)
  10. Microcentrifuge, 4 °C (Eppendorf)
  11. Fisher Scientific Sonic dismembrator (Fisher Scientific, model: 100 )
  12. Vortex (Thermo Fisher Scientific, Thermo ScientificTM, model: LP Vortex Mixer , catalog number: 88880018)

Procedure

  1. Growing and maintaining Caco-2 cell lines
    1. Using MF-Millipore filters prepare media, Caco-2, human colon adenocarcinoma cells are maintained in cEMEM (complete EMEM, see Recipes)
    2. Seeding cells:
      1. Thaw a vial of frozen Caco-2 cells from ATCC (or previously frozen cells in the lab) by gentle agitation in a 37 °C water bath. To reduce the possibility of contamination, keep the O-ring and cap of the tube out of the water. Thawing should be rapid (approximately 2 min)
      2. Remove the vial from the water bath as soon as the contents are thawed, and decontaminate by dipping in or spraying with 70% ethanol. All of the operations from this point on should be carried out under strict aseptic conditions.
      3. Transfer the vial contents to a centrifuge tube containing 9.0 ml cEMEM and spin at 125 x g for 5 to 7 min. Discard supernatant. Resuspend cells in 1 ml cEMEM and transfer the cell suspension to flask containing pre-warmed (37 °C) cEMEM. Place the flask in CO2 forced-air incubator at 37 °C.
      4. 24 h after seeding, check the cells under microscope for attachment, if attached to flask surface, slowly aspirate media to remove dead cells and add pre-warmed (37 °C) cEMEM.
    3. Re-feed cells every 48-72 h or when the color of the media changes. For re-feeding the cells, carefully aspirate the existing media using pipette and add fresh media to the flasks without disturbing the cells.
    4. Check cells for contamination and confluency every 48 h. When 80% of the flask surface is covered by cells, they are ready for passaging (Natoli et al., 2012).Cells need to be passaged for a minimum of 3 cycles before using them for the experiment. When the flask is contaminated the media is cloudy and cells do not adhere to the flask surface.
    5. For passaging, remove media and wash monolayer with 1x PBS. Add 2-4 ml (depending on the size of flask) of trypsin-EDTA solution and incubate at 37 °C for 1-4 min (do not over incubate cells). When cells start sloughing add media (1:10, trypsin to media ratio) to inactivate trypsin and thoroughly pipette several times to get a uniform cell suspension.
    6. Count cells using a hemacytometer and seed a 12 well cell culture plate with a density of 105 cells/well. Each cell will require 1 to 2 ml depending and the size and manufacturer. Calculate the cells accordingly.

  2. Preparation of bacterial culture
    1. Using sterile microbiology technique streak listeria strains (WT and Mutant) from frozen cultures on BHI agar and place them in a 37 °C incubator for 48 h.
    2. From a freshly grown bacterial culture plate pick single isolated colony with a pipette tip and drop it in a 15 ml culture tube containing 10 ml BHI broth. Incubate the tubes in a 37 °C shaker for overnight. (see Note 5)
    3. Overnight cultures (15 ml) of F2365 (wild type) and F2365∆2117 (mutant) L. monocytogenes strains in BHI broth are adjusted to an OD600 = 1.0 using fresh BHI broth on the day of infection if necessary.
    4. For each strain, pellet 100 µl of the above bacterial suspension in a bench top micro centrifuge for 2 min at 3,000 x g.
    5. Wash pellet and resuspend it in 1 ml of fresh warm (37 °C) cEMEM.
    6. Use 30 µl from step B3 for infections with Caco-2 cells.

  3. Adhesion assay
    1. Plates with Caco-2 cells from step A5 are infected following the template shown in Figure 1 with bacterial culture from step B3 (Multiplicity of infection [MOI]: 1 Caco-2 cell:10 bacterial cells).


      Figure 1. Plate template for infection of Caco-2 cells. Caco-2 cells are seeded in 12 well cell culture plates and infected with appropriate bacterial strain as labeled in the template. Infections were performed in three replicates per plate for each strain. Three wells were uninfected or untreated and three wells were infected with cEMEM sans bacteria and acted as negative controls (NC1 and NC2).

    2. Infect one well at a time and change pipette tip after every infection. (see Note 4)
    3. Centrifuge the plates briefly for 45 sec and incubate them at 37 °C for 30 min. Centrifugation aids mixing and binding of bacteria to mammalian cells.
    4. After incubation wash cells five times with 1x PBS and lyse using 500 µl of cold 0.1% Triton X-100.
    5. The resulting suspensions are diluted (1/100, 1/1,000 and 1/10,000) using 1xPBS.
    6. 100 µl of 1/1,000 and 1/10,000 diluted suspensions are spread on BHI agar, and grown at 37 °C for 48 h. (see Note 5)
    7. 48 h post incubation count bacterial colonies, record and calculate colony forming units.

  4. Invasion assay
    1. Plates with Caco-2 cells from step A5 are infected with bacterial culture from step B3 (MOI:1 Caco-2 cell:10 bacterial cells) as shown in Figure 1.
    2. Infect one well at a time and change pipette tip after every infection. (see Note 4)
    3. Centrifuge the plates briefly for 45 sec and incubate them at 37 °C for 2 h.
    4. After incubation wash twice with 1x PBS, and add fresh media containing gentamycin (100 µg/ml) to kill extracellular bacteria.
    5. At 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 h post-infection, cells are washed twice with 1x PBS and lysed using 500 µl of cold 0.1% Triton X-100. Once lysed cells float in the solution, using a pipette carefully mix the solution (without splashing) to remove adherent cells if any. Pipetting also helps in breaking mammalian cells.
    6. Transfer lysate from each well to separate appropriately labelled microcentrifuge tubes and proceed to sonication.
    7. Sonic dismembrator settings: setting 3 (operation frequency: 20 kHz and power rating: 120 watts), 3 pulses, 5 sec each helps in further lysis of Caco-2 cells but does not harm L. monocytogenes.
    8. 100 µl of 1/1,000 and 1/10,000 diluted suspensions are spread on BHI agar, and grown at 37 °C for 48 h. (see Note 5)
    9. 48 h post incubation count bacterial colonies, record and calculate colony forming units.

Data analysis

Note: Data can be analyzed by performing Student’s t-test using Microsoft excel.

  1. Calculate colony forming units based on the dilutions performed during plating.
  2. For Student’s t-test use excel to calculate P-value (alpha), to investigate significant difference in adherent and intracellular bacterial numbers at post-infection in Caco-2 cells. An alpha’s level of 0.05 was used to determine statistical significance.
  3. Calculate standard errors (for error bars) using Microsoft excel.
  4. Using Microsoft excel plot a bar graph for adhesion assay(Figure 2) and point scatter graph for invasion assay (Figure 3).


    Figure 2. Attachment to Caco-2 cells by F2365 and F2365∆2117. Each bar represents the mean of six replicates; standard error is indicated by error bars. *Indicates statistically significant difference from the other treatments. An alpha level of 0.05 was used to determine statistical significance.


    Figure 3. Invasion of Caco-2 cell line by F2365 and F2365∆2117. Each point represents the mean of six replicates; standard error is indicated by error bars. Statistically significant differences between F2365 and F2365∆2117 are indicated by *. An alpha level of 0.05 was used to determine statistical significance.

Notes

  1. Make a note of dilutions when plating on BHI agar to use later for calculating actual number of bacteria colony forming units from bacterial counts.
  2. Perform all infections in triplicates, and execute 2 independent assays for each strain.
  3. Include negative controls as shown and described in Figure 1. These negative controls should not produce colonies on BHI plates. If colonies are observed in negative controls then there has been a potential contamination and the experiments need to be repeated.
  4. For each time point use one plate. Use two plates for each time point to obtain data for six replicates.
  5. All the steps are done aseptically or in sterile conditions during this experiment, thus only one type of colonies (regular, round bacterial colonies) or no colonies should be observed on the plates. Listerial colonies are regular white rounded colonies occurring singly or in short chains. If there are multiple colored or non-identical colonies then there has been a contamination.

Recipes

  1. cEMEM (filter sterilized)
    400 ml EMEM
    100 ml FBS
    5 ml Pen/Step
  2. 0.1% Triton-X 100 (filter sterilized)
    99.9 ml Milli-Q water
    100 µl 100% Triton-X 100
    3. 1x PBS (filter sterilized)
    90 ml Milli-Q water
    10 ml 10x PBS

Acknowledgments

This protocol was adapted from the previously published studies (Reddy et al., 2016). This project was funded by USDA ARS Agreement #58-6402-2729, which is operated under USDA CRIS project MIS501170, ‘Mississippi Center for Food Safety and Post-Harvest Technology’.

References

  1. Angelis, I. D., and Turco, L. (2011). Caco-2 cells as a model for intestinal absorption. Curr Protoc Toxicol Chapter 20.
  2. Jaradat, Z. W. and Bhunia, A. K. (2003). Adhesion, invasion, and translocation characteristics of Listeria monocytogenes serotypes in Caco-2 cell and mouse models. Appl Environ Microbiol 69(6): 3640-3645.
  3. Lecuit, M. (2005). Understanding how Listeria monocytogenes targets and crosses host barriers. Clin Microbiol Infect 11(6): 430-436.
  4. Natoli, M., Leoni, B. D., D'Agnano, I., Zucco, F. and Felsani, A. (2012). Good Caco-2 cell culture practices. Toxicol In Vitro 26(8): 1243-1246.
  5. Reddy, S., Akgul, A., Karsi, A., Abdelhamed, H., Wills, R. W. and Lawrence, M. L. (2016). The role of Listeria monocytogenes cell wall surface anchor protein LapB in virulence, adherence, and intracellular replication. Microb Pathog 92: 19-25.
  6. Reddy, S. and Lawrence, M. L. (2014). Virulence characterization of Listeria monocytogenes. Methods Mol Biol 1157: 157-165.
  7. Sambuy, Y., De Angelis, I., Ranaldi, G., Scarino, M. L., Stammati, A. and Zucco, F. (2005). The Caco-2 cell line as a model of the intestinal barrier: influence of cell and culture-related factors on Caco-2 cell functional characteristics. Cell Biol Toxicol 21(1): 1-26.

简介

单核细胞增生利斯特氏菌是一种重要的革兰氏阳性食源性病原体,是即食食品中的一个特殊问题。它具有在诸如制冷温度和高盐浓度的恶劣条件下生存的能力,并且已知可以穿过肠,胎盘和血脑屏障。已经使用了诸如子宫颈,肝脏,树突状细胞,肠和巨噬细胞的几种癌细胞系来研究人细胞中李斯特菌的体外扩增和存活。人肠上皮细胞已被用于研究李斯特菌如何穿过肠屏障并引起感染。本文中的方案描述了生长Caco-2细胞的过程,维持细胞并将其用于粘附和侵袭测定。在粘附测定期间,将细胞与李斯特菌孵育30分钟,但是在侵袭测定中,细胞生长在感染后的几个时间点被停止以监测细胞中李斯特菌的生长和存活率。

背景 作为兼性细胞内细菌的单核细胞增生利斯特氏菌可以在吞噬细胞和非吞噬细胞中进入,存活和繁殖,并且细菌的这种性质已被广泛研究和理解。作为食源性病原体,它通过肠屏障通过肠进入人体的血液流。因此,人肠细胞用作体外培养基以研究单核细胞增生利斯特氏菌的粘附和细胞内存活。人结肠腺癌细胞Caco-2细胞被广泛用作肠屏障的模型(Angelis和Turco,2011)。本文所述的方案解释了生长和维持Caco-2细胞的方法,然后感染它们进行研究李斯特菌种的粘附和侵袭性质。对于也用于研究利斯特氏菌的HT-29(人结肠直肠腺癌细胞)和Tc7细胞(Caco-2细胞系的亚克隆)等细胞也可以使用该方案。这些测定通常用于比较李氏体突变体与宽型(WT)菌株的致病性(Reddy和Lawrence,2014)。粘附测定是直接的,其中细菌与Caco-2细胞孵育30分钟,并比较突变体和WT菌株的细菌计数,以观察由于突变导致的粘附性质的任何改变。在入侵测定期间在不同时间点获得的细菌计数提供了关于人类细胞中李斯特菌的存活的更多信息,并且突变体和WT菌株之间的这些计数的比较提供了关于人细胞突变后李斯特氏菌适应性变化的信息。以前已经成功地使用该方案来研究李斯特菌株的粘附和侵袭性质(Jaradat和Bhunia,2003; Lecuit,2005; Sambuy等人,2005; Reddy等人,2016)。

关键字:粘附试验, 侵袭试验, 李斯特菌, Caco-2细胞

材料和试剂

  1. MF-Millipore过滤器(EMD Millipre,目录号:SCGPU05RE)
  2. Eppendorf管(Eppendorf,目录号:022363204)
  3. 细胞培养瓶(Corning,目录号:3275)
  4. 细胞培养板(12孔培养皿接种板,无菌)(EMD Millipre,目录号:PIMWS1250)
  5. 移液器尖端
  6. 15 ml培养管
  7. 电池刮板(Corning,目录号:3010)
  8. ZapCap瓶盖过滤器,孔径0.2μm(Maine Manufacturing,目录号:10443421)
  9. 血清学移液器(1,5,10,25,50 ml)(康宁,目录号:4010,4050,4100,4250,4501)
  10. 培养管(Sigma-Aldrich,目录号:T1661)
  11. 单核细胞增多性李斯特菌菌株F2365(野生型菌株)和F2365Δ2117(突变菌株)(Reddy等人,2016)
  12. Caco-2细胞系(ATCC,目录号:HTB-37)
  13. 70%乙醇(Fisher Scientific,目录号:BP82014)
  14. 胰蛋白酶-EDTA溶液(Sigma-Aldrich,目录号:T3924)
  15. 脑心脏输注(BHI)琼脂(Sigma-Aldrich,目录号:70138)
  16. BHI肉汤(Sigma-Aldrich,目录号:53286)
  17. 庆大霉素(Sigma-Aldrich,目录号:G1397)
  18. 鹰的最低必需培养基(EMEM)(ATCC,目录号:30-2003)
  19. 胎牛血清,认证,热灭活(FBS)(Thermo Fisher Scientific,Gibco TM,目录号:10082147)
  20. 青霉素 - 链霉素(10,000U/ml)(Thermo Fisher Scientific,Gibco TM,目录号:15140122)
  21. Dulbecco的磷酸盐缓冲盐水(Sigma-Aldrich,目录号:D8537)
  22. Triton X-100(Sigma-Aldrich,目录号:234729)
    注意:本产品已停产。
  23. 磷酸盐缓冲盐水,10倍(PBS)(Sigma-Aldrich,目录号:P5493)
  24. cEMEM(见配方)
  25. 0.1%Triton-X 100(参见食谱)
  26. 1x PBS(见食谱)

设备

  1. 水浴,37°C(Thermo Fisher Scientific)
  2. CO 2/2强制空气培养箱(Thermo Fisher Scientific,Thermo Scientific TM,型号:Forma TM Steri-Cult CO 2 孵化器,目录号:3307TS)
  3. 生物安全柜(Thermo Fisher Scientific,Thermo Scientific TM,型号:1300系列II类)
  4. 倒置显微镜(Nikon Instruments,型号:Eclipse Ti-S)
  5. 移液器(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:4642070)
  6. Pipettor(Daigger Scientific,型号:便携式移液器XP移液器控制器)
  7. 血细胞计数仪(Fisher Scientific,目录号:S17040)
  8. 37℃培养箱(Thermo Fisher Scientific)
  9. 37℃振荡器(Thermo Fisher Scientific)
  10. 微量离心机,4°C(Eppendorf)
  11. 费雪科学声波分解器(Fisher Scientific,型号:100)
  12. 涡旋(Thermo Fisher Scientific,Thermo Scientific TM,型号:LP Vortex Mixer,目录号:88880018)

程序

  1. 生长和维持Caco-2细胞系
    1. 使用MF-Millipore过滤器制备培养基,将Caco-2,人结肠腺癌细胞维持在cEMEM中(完整的EMEM,参见食谱)
    2. 播种细胞:
      1. 通过在37℃水浴中温和搅拌,从ATCC(或实验室中先前冷冻的细胞)中解冻一瓶冷冻的Caco-2细胞。为了减少污染的可能性,请将O型圈和管帽盖保持在水中。解冻应快速(约2分钟)
      2. 一旦内容物解冻,就从水浴中取出小瓶,并通过浸入或喷洒70%乙醇来净化。从这一点开始的所有操作都应该在严格的无菌条件下进行
      3. 将小瓶内容物转移到含有9.0ml cEMEM的离心管中,并以125×g旋转5至7分钟。丢弃上清液。将细胞重悬于1ml cEMEM中,并将细胞悬浮液转移到含预热(37℃)cEMEM的烧瓶中。将烧瓶置于CO 2/2强制空气培养箱中,37℃
      4. 接种后24小时,检查显微镜下的细胞附着,如果连接到烧瓶表面,缓慢吸出培养基以除去死细胞并加入预热(37℃)的cEMEM。
    3. 每48-72小时或当媒体的颜色发生变化时重新进入细胞。为了重新喂养细胞,用吸管小心吸出现有培养基,并将新鲜培养基添加到烧瓶中,而不会干扰细胞。
    4. 每48小时检查一次细胞的污染和汇合。当烧瓶表面的80%被细胞覆盖时,它们可以进行传代(Natoli等人,2012)。在将它们用于实验之前,需要传递至少3个周期的细胞。当烧瓶被污染时,介质浑浊,细胞不粘附到烧瓶表面
    5. 对于传代,移除介质并用1x PBS洗涤单层。加入胰蛋白酶-EDTA溶液2-4ml(取决于烧瓶的大小),并在37℃孵育1-4分钟(不要过孵育细胞)。当细胞开始呕吐时,添加培养基(1:10,胰蛋白酶至培养基比例)以灭活胰蛋白酶并彻底移液数次以获得均匀的细胞悬浮液。
    6. 使用血细胞计数器计数细胞并种子密度为10 5个细胞/孔的12孔细胞培养板。每个电池将需要1到2毫升的尺寸和制造商。相应地计算单元格。

  2. 细菌培养物的制备
    1. 使用无菌微生物学技术的条纹李斯特菌菌株(WT和Mutant)从BHI琼脂上的冷冻培养物中,并将它们置于37℃的培养箱中48小时。
    2. 从新鲜生长的细菌培养板中,用移液管尖端挑取单个分离的菌落,并将其放入含有10ml BHI肉汤的15ml培养管中。将试管在37℃振荡器中孵育过夜。 (见注5)
    3. F2365(野生型)和F2365Δ2117(突变体)L的夜间培养物(15ml)。如果需要,在BHI肉汤中的单核细胞增生李斯特氏菌菌株在感染当天使用新鲜BHI肉汤调整至OD 600> 1.0。
    4. 对于每个菌株,将100μl上述细菌悬浮液在台式微型离心机中以3,000xg 沉淀2分钟。
    5. 洗涤沉淀,并将其重悬于1ml新鲜温热(37℃)cEMEM中。
    6. 使用步骤B3使用30μl感染Caco-2细胞。

  3. 粘附分析
    1. 使用来自步骤B3的细菌培养物(感染的多重性[MOI]:1Caco-2细胞:10个细菌细胞),按照图1所示的模板感染来自步骤A5的具有Caco-2细胞的平板。


      图1.用于感染Caco-2细胞的板模板。将Caco-2细胞接种在12孔细胞培养板中,并用模板中标记的合适的细菌菌株感染。对于每个菌株,以平板每3次重复进行感染。三口井未感染或未经处理,三口井感染无细胞生长激素免疫细胞,作为阴性对照(NC1和NC2)。

    2. 一次感染一口,每次感染后更换移液管。 (见注4)
    3. 将板短暂离心45秒,并在37℃孵育30分钟。离心有助于细菌与哺乳动物细胞的混合和结合
    4. 孵育后,用1x PBS洗涤细胞5次,用500μl冷的0.1%Triton X-100溶解
    5. 使用1xPBS将所得悬浮液稀释(1/100,1/1,000和1/10,000)。
    6. 将100μl1/1,000和1/10,000稀释的悬浮液铺在BHI琼脂上,并在37℃下培养48小时。 (见注5)
    7. 孵化后48小时计数细菌菌落,记录并计算菌落形成单位
  4. 入侵检测
    1. 来自步骤A5的具有Caco-2细胞的板用来自步骤B3(MOI:1Caco-2细胞:10个细菌细胞)的细菌培养物感染,如图1所示。
    2. 一次感染一口,每次感染后更换移液管。 (见注4)
    3. 将板短暂离心45秒,并在37℃孵育2小时
    4. 孵育后用1x PBS洗涤两次,加入含有庆大霉素(100μg/ml)的新鲜培养基,以杀死细胞外细菌。
    5. 在感染后2,4,6,8,10,12,14,16,18,20,22和24小时,用1x PBS洗涤细胞两次,并使用500μl冷的0.1%Triton X-100 。一旦裂解的细胞漂浮在溶液中,使用移液管小心地混合溶液(无飞溅)去除附着细胞(如果有的话)。移液也有助于破坏哺乳动物细胞。
    6. 从每个孔转移裂解物,分离适当标记的微量离心管,并进行超声处理
    7. 声波除垢器设置:设置3(操作频率:20 kHz和额定功率:120瓦),3个脉冲,5秒每个有助于进一步裂解Caco-2细胞,但不会伤害L。单核细胞增生素
    8. 将100μl1/1,000和1/10,000稀释的悬浮液铺在BHI琼脂上,并在37℃下培养48小时。 (见注5)
    9. 孵化后48小时计数细菌菌落,记录并计算菌落形成单位

数据分析

注意:可以使用Microsoft Excel执行学生的t检验来分析数据。

  1. 根据电镀过程中的稀释度计算菌落形成单位
  2. 对于Student's t 测试,使用excel来计算P 值(alpha),以调查Caco-2细胞感染后的粘附和细胞内细菌数量的显着差异。使用0.05的α水平来确定统计学显着性
  3. 使用Microsoft Excel来计算标准错误(用于错误栏)。
  4. 使用Microsoft Excel绘制粘附分析的条形图(图2)和入侵测定的点散点图(图3)。


    图2. F2365和F2365Δ2117附着于Caco-2细胞。每个条代表六次重复的平均值;标准误差由误差线指示。 *与其他治疗有显着差异。使用0.05水平的α水平来确定统计学显着性

    图3. F2365和F2365Δ2117侵袭Caco-2细胞系。每个点代表六次重复的平均值;标准误差由误差线指示。 F2365和F2365Δ2117之间的统计学显着性差异由*表示。使用0.05水平的α水平来确定统计学显着性

笔记

  1. 记录BHI琼脂上的稀释液,以便稍后使用,从细菌计数计算细菌菌落形成单位的实际数量。
  2. 执行所有感染一式三份,并对每个菌株执行2次独立测定。
  3. 包括如图1所示和描述的阴性对照。这些阴性对照不应在BHI平板上产生菌落。如果在阴性对照中观察到菌落,那么就有潜在的污染,需要重复实验
  4. 对于每个时间点使用一个板。每个时间点使用两个平板,以获取六个重复的数据。
  5. 所有步骤都是无菌或无菌条件下进行的,因此在板上只能观察到一种类型的菌落(常规圆形细菌菌落)或没有菌落。利斯特里克菌落是单个或短链发生的常规白色圆形菌落。如果有多个有色或非相同的菌落,那么就会有污染。

食谱

  1. cEMEM(过滤灭菌)
    400 ml EMEM
    100毫升FBS
    5毫升笔/步骤
  2. 0.1%Triton-X 100(过滤灭菌)
    99.9毫升Milli-Q水
    100μl100%Triton-X 100
    3. 1x PBS(过滤灭菌)
    90 ml Milli-Q水
    10ml 10倍PBS

致谢

该协议是从以前发表的研究(Reddy等人,2016)改编的。该项目由USDA ARS协议#58-6402-2729资助,该协议由USDA CRIS项目MIS501170"密西西比州食品安全和收获后技术中心"运营。

参考

  1. Angelis,ID和Turco L.(2011)。  Caco-2细胞作为肠道吸收的模型。 Curr Protoc Toxicol 第20章。
  2. Jaradat,ZW和Bhunia,AK(2003)。  粘合力,Caco-2细胞和小鼠模型中单核细胞增生利斯特氏菌血清型的入侵和易位特征。应用环境微生物69(6):3640-3645。 br />
  3. Lecuit,M。(2005)。  了解如何>单核细胞增生利斯特氏菌靶向并穿过主体障碍。 Clin Microbiol Infect 11(6):430-436。
  4. Natoli,M.,Leoni,BD,D'Agnano,I.,Zucco,F.and Felsani,A。(2012)。< a class ="ke-insertfile"href ="http://www.ncbi .nlm.nih.gov/pubmed/22465559"target ="_ blank">良好的Caco-2细胞培养实践 体外毒素 26(8):1243-1246。 />
  5. Reddy,S.,Akgul,A.,Karsi,A.,Abdelhamed,H.,Wills,RW和Lawrence,ML(2016)。  单核细胞增生利斯特氏菌细胞壁表面锚蛋白LapB在毒力,粘附和细胞内复制中的作用。 Microb Pathog em> 92:19-25。
  6. Reddy,S.and Lawrence,ML(2014)。  单核细胞增生利斯特氏菌的毒力表征。方法Mol Biol 1157:157-165。
  7. Sambuy,Y.,De Angelis,I.,Ranaldi,G.,Scarino,ML,Stammati,A.and Zucco,F。(2005)。< a class ="ke-insertfile"href ="http: /www.ncbi.nlm.nih.gov/pubmed/15868485"target ="_ blank">作为肠屏障模型的Caco-2细胞系:细胞和培养物相关因子对Caco-2细胞功能特征的影响细胞生物毒素21(1):1-26。
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引用:Reddy, S. and Austin, F. (2017). Adhesion and Invasion Assay Procedure Using Caco-2 Cells for Listeria monocytogenes. Bio-protocol 7(9): e2267. DOI: 10.21769/BioProtoc.2267.
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