Mccoy s 5a modified medium

HT-29 cells were grown as previously described [11 (link)] and maintained in McCoy′s 5A modified medium (Merck) supplemented with 10% fetal bovine serum (FBS) using 75 cm² tissue culture flasks incubated at 37 °C in 5% CO₂ in a humidified atmosphere. Once the cells were nearing confluency (approximately 80%–90%), they were passaged into 48 well tissue culture plates (Sarstedt Ltd., Wexford, Ireland) at a density of 1 × 10⁵ cells/mL between passages 15–21. The cells were then used once fully confluent (approximately 2 × 10⁶ cells/well). The media was changed every other day and supplemented with 2% FBS 24 h prior to use.

Scanning electron microscopy (SEM) was employed to visualise the attachment of Bifidobacterium cells to IgG treated HT-29 cells versus untreated HT-29 cells. For adhesion assays, HT-29 cells (approximately 1 × 10⁵ cells/mL) were seeded in 12-well tissue culture plates (Merck) on microscopy cover glasses in McCoy’s 5A modified medium (Merck) and incubated at 37 °C in a humidified atmosphere of 5% CO2, until reaching 90 to 100% confluency. HT-29 cells were exposed to 16 mg/mL colostral IgG as described previously. Non-supplemented McCoy’s 5A medium was used as a non-treated control (NT). The adhesion assay was performed as previously described above. After 2 h of incubation with B. longum 8809, the cells were then observed by Scanning Electron Microscopy (SEM) using the method of Khokhlov et al. (2012 ). The HT-29 monolayer was washed three times with 1 mL PBS and fixed with 4% (w/v) glutaraldehyde (Merck), in 0.1 M phosphate (PB) buffer, (pH 6.8–7.4) for 1 h at room temperature. After washing three times with PBS, the HT-29 monolayer was dehydrated in a graded ethanol series (25% v/v, 50% v/v, 75% v/v, 95% v/v and 100% v/v). The cells were air dried and sputter coated with chromium gold (Emitech K550X, Ashford, UK). The microscope cover glasses were then examined with a Field Emission Scanning Electron Microscope at 7 kV (FE-SEM; Zeiss Supra Gemini, Darmstadt, Germany).