Chapter 2 Materials and Methods
2.2 Intestinal layer components
2.2.1 Mucin
2.2.1.1 Sepharose beads coated with mucin
Porcine MUC2 mucin (Sigma Aldrich, Auckland, New Zealand) was used for the in vitro
binding assay. The crude mucin was coupled to epoxy-activated Sepharose 6B (GE Healthcare, Auckland, New Zealand) as described by Alvarez et al. [460] and according to manufacturer’s instructions. A negative control medium was also prepared (ethanol-amine (EtOH-amine) blocked beads with no mucin).
In brief, MUC2 was hydrated in borate buffer (0.05 M, pH 9.0). pH 9 was chosen as a balance between an increased reactivity of the epoxy groups at higher pH values and a physiological environment for the mucin. Epoxy-Sepharose was swollen in ultrapure (MilliQ) water. Sepharose was washed with at least 200 ml MilliQ water per 1 mg beads. Washed Sepharose and hydrated MUC2 in borate buffer were mixed and incubated over night at 90 revolutions.min-1 (rpm) and 25°C. The next day, the beads were washed with coupling buffer to remove unbound mucin and 1 M EtOH-amine (Sigma Aldrich) was added to block yet unbound epoxy groups. Incubation was at 90 rpm and 45°C overnight. Finally, the beads were washed with three cycles of alternating 0.1 M acetate buffer (pH 3.8, 0.5 M sodium chloride (NaCl, LabServ Pronalys, Thermo Fisher Scientific, Auckland, New Zealand)) and 0.1 M Tris-HCl buffer (pH 8.2, 0.5 M NaCl) to ensure that no weakly attached (ionically) ligand remained bound to the immobilised mucin. The ready MUC2 covered beads were stored in borate buffer and 25% ethanol (EtOH, Sigma Aldrich)) at 4°C until use.
The negative control beads were prepared the same way, but without the addition of MUC2. The swollen and washed Sepharose beads were directly incubated with 1 M EtOH-amine, followed by the wash cycle. The beads were stored in MilliQ water and 25% EtOH at 4°C until use.
61
2.2.1.2 Testing mucin coverage
Confocal Laser Scanning Microscopy
To visualise the mucin covering the Sepharose beads, the MUC2 was labelled using a lectin- flurophore conjugate. Labelled mucins were then evaluated using a confocal laser scanning microscope.
AlexaFluor488 labelled WGA (Sigma Aldrich), a lectin which binds to mucin, was dissolved at 1 mg.ml-1 in phosphate buffered saline (PBS) and aliquoted to avoid repeated freeze-thaw- cycles. Unused label was stored at -20°C. Next, 100 μl MUC2 covered beads or negative control beads were transferred into tubes. The beads were washed three times with PBS-T (0.05% Tween20 (BioRad, Auckland, New Zealand) in PBS) to remove the storage buffers. Next the beads were blocked with 1% bovine serum albumin (BSA, ICPbio, Auckland, New Zealand) in PBS for 1 hr at room temperature. Again, the beads were washed three times with PBS-T before adding 100 μl lectin solution at different concentrations (0.1 to 10 μg.ml-1 lectin) to the beads. This was followed by another 1 hr incubation at room temperature and three washes with PBS- T. Samples were kept in the dark until use. For analysis the beads were transferred onto slides and a coverslip was sealed with nail polish. For visualisation an Olympus Fluoview FV1000 confocal laser scanning microscope and Olympus Fluoview v1.7a software (Olympus America Inc., Center Valley, PA, USA) were used.
PBS was made from a 10-times stock solution. Therefore 80.0 g NaCl, 2.0 g KCl (Sigma Aldrich), 14.4 g Na2HPO4 (Fisher Chemical, Thermo Fisher Scientific, Auckland, New Zealand) and 2.4 g KH2PO4 (BDH AnalaR, VWR International Ltd., Auckland, New Zealand) were dissolved in 700 ml MilliQ water. After the pH was adjusted to 7.4 with HCl (Fisher Chemical, Thermo Fisher Scientific, Auckland, New Zealand) and sodium hydroxide (NaOH, Fisher Chemical), the volume was topped up to 1000 ml with MilliQ water.
62
Measurement of mucin using a 2-D Quant Kit
The amount of mucin in the binding buffer before and after coupling to Sepahrose beads was measured with 2-D Quant Kit (GE Healthcare) according to manufacturer’s instructions. All regaents and solutions were contained in the kit. In brief, mucin was precipitated with the provided reagents, and re-dissolved in a copper solution. After adding a colour reagent, the amount of protein in the samples was measured against a BSA standard curve by absorption at 480 nm [461].
Total combustion (LECO)
Determination of total nitrogen by total combustion (LECO) was performed at the Riddet Institute (Palmerston North, New Zealand) following the method described in AOAC 968.06. Thereby, nitrogen in the sample was freed through pyrolysis followed by combustion and swept into a nitrometer (with CO2 as carrier) where residual nitrogen was measured. Total nitrogen was converted to net protein through a conversion factor of 6.25, employing Equation 1.
(Eq. 1)
Measurement of FITC-WGA binding to mucin using a plate reader
For the colorimetric mucin quantification, AlexaFluor488 labelled WGA (WGA488) was used to indirectly detect the glycoprotein. The labelling was performed as described for confocal laser scanning microscopy. The samples analysed were MUC2 beads with label. Further, controls were prepared: MUC2 beads without label, EtOH-amine blocked beads with label, EtOH-amine blocked beads without label, pure beads with label, pure beads without label.
Alcian Blue (periodic acid Schiff)
Alcian blue (AB, Sigma Aldrich) was used to visualise the mucin before it was coupled to the Sepharose beads and thus allow tracking after coupling. Mucin was hydrated in MilliQ water for at least 5 hr before adding 100 μl AB solution (several grains AB in 3% aq acetic acid (AnalaR)). Double strength borate buffer (0.1 M, pH 9.0) was added (1:1 volume ratio to MilliQ water) to the mix to get a normal strength binding buffer. Epoxy-Sepharose was prepared as
63
described in Section 3.1.1.1, mixed with the AB-mucin binding-buffer and incubated at 25°C overnight and 90 rpm. Distribution of the blue colour, indicating mucin, was determined visually and used to assess the degree of binding.
2.2.2 Bacteria
Bacterial strains were kindly provided by Dr U. Sonnenborn, Ardeypharm GmbH, Germany (E. coli Nissle 1917) and Prof. G. Pier, Harvard Medical School, USA (S. epidermidis 1457 and S. epidermidis 1457 M10). S. epidermidis 1457 M10 is an icaADBC knock-out which does not
produce pGlcNAc. The stocks were plated onto Luria broth (Invitrogen, Thermo Fisher Scientific, Auckland, New Zealand) agar plates and incubated at 37°C. Once colonies formed, a single colony each was transferred into liquid culture medium; Luria broth Lenox L broth (Invitrogen) for E. coli Nissle 1917 and tryptic soy broth (BD BactoTM, BD, Auckland, New
Zealand) for S. epidermidis 1457 and S. epidermidis 1457 M10. Cultures were grown overnight
at 37°C and 180 rpm in a shaking incubator (Infors HT, Ecotron, Total Lab Systems Ltd. Auckland, New Zealand). To prepare glycerol stocks, 500 μl culture was mixed with 500 μl glycerol (BDH AnalaR) and stored at -80°C until use.
2.2.3 Intestinal epithelial cells
2.2.3.1 Mono cultures
Caco-2 cells were obtained from ATCC HTB-37 and used from passages 56 to 59 and 40 to 43. HT29 cells were obtained from ATCC HTB-38, adapted to 10-7M MTX (Dr R. Anderson, AgResearch Grasslands, New Zealand) and used from passages 14 to 24.
Caco-2 cells were grown in Dubelco’s Modified Eagle Medium (DMEM, Invitrogen) with 10% foetal calf serum (FCS, Gibco, Thermo Fisher Scientific, Auckland, New Zealand or Moregate, Bulimba, Australia). HT29-MTX cells were grown in McCoys medium (Gibco) with 10% FCS and 1% non-essential amino acids (Life Technologies, Thermo Fisher Scientific, Auckland, New Zealand). Cells were grown at 37°C in 5% carbon dioxide and culture medium was changed at least every third day. After reaching confluence, cells were detached with TriplE
64
Express (Life Technologies). After detachment, DMEM was added to neutralise the TriplE and the cells were pelleted by centrifugation (5 min, 103 rpm). After re-suspending, 100 μl of the cell suspension was mixed with 900 μl warmed Crystal Violet (Gibco) and counted in a haemocytometer. The remaining cell suspension was re-seeded at 4 x 104 cells.cm-2. In preparation for the adhesion assay, cells were grown in 24-well plates (Thermo Scientific Nunc™, Thermo Fisher Scientific, Auckland, New Zealand) for 16 to 18 days. All cell lines were grown on DMEM with 10% FCS and 1% non-essential amino acids to avoid differences in treatment between mono- and co-cultures. HT29 can produce mucin in DMEM with 10% FCS [462, 463].
2.2.3.2 Co-culture
After reaching confluence, mono-cultures were detached and cells counted as described in Section 2.2.3.1. The cells were gently mixed to the desired ratio (e.g. 90% Caco-2 and 10% HT29-MTX) and seeded at 4 x 104 cells.cm-2 in DMEM with 10% FCS and 1% NEAA. Co- culture medium was changed at least every third day.