The immune stimulating ability of each sample was determined by measuring the induction of an immune response in peripheral blood mononuclear cells (PBMCs) by the sample. This response was detected and quantified using a cytometric bead array (CBA) technique to measure cytokines secreted by the blood cells.
2.2.8.1Limulus Amoebocyte Lysate (LAL) Assay
The Limulus Amoebocyte Lysate (LAL) assay was used to determine the amount of any contaminating lipopolysaccharide (LPS) in samples prior to testing for immune activity, primarily to confirm that samples to be tested did not contain sufficient LPS to induce a detectable immune response. The assay was carried out in a sterile microplate according to the supplier’s instructions, except that the volumes of sample, lysate, chromogenic substrate and stop reagent (25 % v/v glacial acetic acid) were reduced to 20, 20, 40 and 40 μL, respectively. The absorbance at 405 nm was read in a Molecular Devices OPTImax™ tuneable microplate reader. Samples were tested in duplicate. The detection range for this assay is 0.1 – 1.0 Endotoxin units per millilitre (EU/mL).
2.2.8.2PBMC Assay
PBMCs are a subset of blood cells including a number of different types of immune cells. These cells provide a convenient method of measuring immune responses. When examining the immune responses to probiotics, the immune response in the intestinal tract is often considered to be the most relevant. While the immune response in the blood may not necessarily approximate that in the gut exactly, the same major immune cell types are represented.
Cell culture media was prepared by filter-sterilising a freshly made solution of RPMI 1640 + GlutaMAX™ containing 10 % v/v fetal bovine serum (FBS) and a
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penicillin/streptomycin mixture (at final concentrations of 100 U and 100 μg per mL, respectively). A 1 mL vial of PBMC stored in liquid nitrogen was quickly thawed, then resuspended gently in 25 mL of culture media prewarmed to 37 ºC. Cells were then centrifuged at 200 ×g for 15 minutes at room temperature before being resuspended in 10 mL of media. At this point a 20 μL aliquot was stained with Trypan blue and counted on a haemocytometer to assess viability. The remaining PBMC suspension was diluted with culture media to give a concentration of 1.1 × 106 viable cells/mL, and 180 μL pipetted into each well of sterile flat-bottomed microplates. Thus after addition of 20 μL of control or sample, each well contained PBMCs at a final concentration of 1.0 × 106 viable cells/mL in all wells, in a final volume of 200 μL.
Samples to be tested in this assay had previously been resuspended or diluted as required into phosphate buffered saline (PBS) and tested for the presence of LPS using the LAL assay (section 2.2.8.1). All samples free from LPS were then mixed thoroughly by vortexing before addition of duplicate or triplicate 20 μL aliquots of each sample to wells containing the PBMCs. Wells testing whole bacterial cells were prepared so that the final concentration of bacteria was 1.0 × 106 cfu/mL. Controls were included with the assay containing final concentrations of each of the following: PBS from the same batch used to prepare the samples (negative control), 1 ȝg/ml bacterial lipopolysaccharide (positive control), a combination of 25 ng/ml phorbol 12-myristate 13-acetate (PMA) with 1 ȝg/ml ionomycin (T cell mitogens, positive control for TNF, IFN-γ and other cytokines), and 10 μg/mL phytohemagglutinin (PHA-P) (also a T cell mitogen known to induce cytokine secretion in PBMCs). The plates were incubated in 5 % CO2 at 37 ºC for 24 hours, after which they were sealed and stored at -80 ºC until
analysed.
2.2.8.3CBA Analysis
The concentrations of cytokines secreted into the cell culture supernatants by the PBMCs were determined using the BD™ CBA Assay Flex Set kits in conjunction with analysis on a BD™ FACSArray™ Bioanalyser. The CBA analysis was performed according to the supplier’s instructions for the BD™ CBA Flex Set kits, the BD™ Soluble Protein Master Buffer Kit and the manuals for the BD™ FACSArray™ instrument, with the following modifications. The volumes of sample/standard, capture
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beads and PE detection reagent were reduced from 50 μL per well to 25 μL per well. Each sample well was resuspended in 100 μL of wash buffer in the final step before analysis on the FACSArray™. For the acquisition settings in the FACSArray™ software controlling the instrument, the number of mixes was changed to three, and the sample volume was increased to 40 μL.
Where possible, each PBMC plate was analysed for all relevant cytokines simultaneously. One notable exception is the analysis of IL-8, which was expressed in PBMC at levels beyond the range of the IL-8 standards, and had to be analysed separately after dilution of the samples in the CBA sample diluent. The number of freeze-thaw cycles of the PBMC plates was kept to as few as possible to minimise the denaturation of cytokine proteins prior to analysis.
After CBA analysis, each PBMC plate was checked for differences across the plate, particularly at the edges, which may be caused by issues such as uneven temperature gradients across a plate during incubation (for example, by plotting cytokine data for every set of twelve wells across the plate). No such effects were observed in this study.