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

Swetha Reddy; Frank  Austin

doi:10.21769/BioProtoc.2267

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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

SR Swetha Reddy email

FA Frank Austin

发布: 2017年05月05日第7卷第9期 DOI: 10.21769/BioProtoc.2267 浏览次数: 15490

评审: Valentine V TrotterSofiane El-Kirat-ChatelAlexander B. Westbye

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The authors used this protocol in:

Cover of Microbial Pathogenesis, featuring study using the protocol.

Mar 2016

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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 foodborne 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 wherein 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

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

Equipment

Water bath, 37 °C (Thermo Fisher Scientific)
CO₂ forced-air incubator (Thermo Fisher Scientific, Thermo Scientific^TM, model: Forma^TM Steri-Cult^TM CO₂ Incubators , catalog number: 3307TS)
Biological safety cabinet (Thermo Fisher Scientific, Thermo Scientific^TM, model: 1300 Series Class II )
Inverted microscope(Nikon Instruments, model: Eclipse Ti-S )
Pipette (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 4642070 )
Pipettor (Daigger Scientific, model: Portable Pipet-Aid XP Pipette Controller )
Hemacytometer (Fisher Scientific, catalog number: S17040 )
37 °C incubator (Thermo Fisher Scientific)
37°C shaker (Thermo Fisher Scientific)
Microcentrifuge, 4 °C (Eppendorf)
Fisher Scientific Sonic dismembrator (Fisher Scientific, model: 100 )
Vortex (Thermo Fisher Scientific, Thermo Scientific^TM, model: LP Vortex Mixer , catalog number: 88880018)

Procedure

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如何引用

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.