General Protocol

Culture Conditions

The IPEC-J2 cells are cultured in DMEM/F-12 mix (Dulbecco's Modified Eagle Medium, Ham's F-12 mixture) and supplemented with HEPES, fetal bovine serum (FBS) or porcine serum (PS), insulin/transferrin/selenium (ITS), penicillin/streptomycin and cultivated in a humid environment at 37 °C with 5 % CO2. The IPEC-J2 cells are usually grown for 1–2 weeks before initiating an experiment. When studying TEER and permeability, IPEC-J2 cells are commonly seeded (1 × 105 cells/well) at confluence in a 'Boyden chamber' insert (upper chamber, apical) on a polyethylene terephthalate (PET) membrane (1.12 cm2, pore size 0.4 μm) in a 12-well plate (lower chamber, basolateral). Cells are seeded (1 × 105/well) in a 12-well plate (flat bottom) to investigate the intracellular oxidative stress and wound healing capacity. Cells are seeded (0.5 × 104 cells/well) in a 96-well plate (flat bottom) to assess viability. The IPEC-J2 cell line is an easy to use and robust cell line, exhibiting structural and functional differentiation pattern characteristics of mature enterocytes (Geens and Niewold 2011).

Experimental Readout

An increasing number of studies use IPEC-J2 cells to investigate interactions of various animal and human pathogens, including Salmonella enterica and pathogenic Escherichia coli (Boyen et al. 2009; Veldhuizen et al. 2009). Pathogenic permeation (e.g. E. coli) is usually presented as colony forming units (CFU). IPEC-J2 cells have also been employed as an initial screening tool for adhesiveness and antiinflammatory properties of potential probiotic microorganisms. IPEC-J2 cells were also used to investigate the effect of prebiotics on the adhesion of probiotic bacterial strains to these cells. Addition of 200 mM calcium has been shown to increase adhesion (Marcinakova et al. 2010), while magnesium and zinc ions had no influence (Larsen et al. 2007). Innate immune responses (e.g. increase in porcine β-defensin 1 and 2 gene expression) in relation to environmental stimuli (e.g. diet or infection)

are investigated with relevance for human and porcine intestinal diseases, specifically in newborns (Schierack et al. 2006; Burkey et al. 2009; Veldhuizen et al. 2009). Numerous studies used IPEC-J2 cells to investigate feedstuffs and antioxidants in relation to inflammation, intestinal permeability and wound healing capacity (Hermes et al. 2011; Ma et al. 2012; Pan et al. 2013; Vergauwen et al. 2015).

Permeability is expressed using TEER, either presented as absolute values or as percentages of control or time point (TEERtx/TEERt0). The net value of the TEER (Ω × cm2) needs to be corrected for background resistance by subtracting the contribution of the cell-free filter and the medium (80–150 Ω × cm2). Alternatively fluorescein isothiocyanate (FITC)-dextran of 4 kDa (FD-4) permeability can be used to indicate monolayer integrity. FD-4 permeability results are presented as a percentage of control or as absolute quantities or concentrations (e.g. picomoles).

Viability and cytotoxicity are most commonly analyzed using the neutral red method, lactate dehydrogenase release or the MTT reduction assay and presented as percentages of control or absorbance values (Table 12.1).

Sample Preparation

It is important only to incubate sterile filtered samples on the IPEC-J2 cells. Otherwise, interpretation of the results will be ambiguous as the effect cannot be contributed to an impurity, a pathogen or the agent of interest.

Samples that are not readily dissolved in water can be dissolved in ethanol or DMSO. The effect of different concentrations of ethanol or DMSO for either 1 or 18 h on the viability of IPEC-J2 cells was investigated (Fig. 12.2). Results show that short and long term incubation do not favor DMSO or ethanol at concentrations below 1 %. DMSO is favored for long-term incubation at concentrations above 1 %. However, concentrations exceeding 1 % are not recommended. Furthermore, it is always important to minimize the concentration of DMSO or ethanol when solubilizing a compound.

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