BioPlex analysis

A Bio-Rad BioPlex 200 system was used in conjunction with a BioPlex Pro Human Diabetes 12 + 2-Plex Panel Complete Kit (catalogue number 171-A4S01M), accord- ing to the supplier’s recommendations, to measure 14 cytokines in human plasma samples (detailed in Table 2.2). Samples were prepared separately for the 12-plex

Metabolite Description

Adipsin Also known as Factor D, found in high levels in adipose (fat) tissue, plays a role in humoral suppression of infectious agents. The level of adipsin is elevated in obese individuals.

Adiponectin Involved in regulating glucose levels (decreases gluconeogenesis and in- creases glucose uptake) as well as the breakdown of fatty acids. Exclu- sively secreted from adipose tissue, highly abundant in plasma.

C-Peptide Produced in a long chain attached to insulin. When cleaved apart, C-peptide plays a role in intracellular signalling pathways, has anti- inflammatory e↵ects as well as aiding in the repair of smooth muscle cells. It is used to distinguish between type I and type II diabetes. GIP Glucose-dependent insulintropic peptide is a member of the secretin fam-

ily of hormones, believed to induce insulin secretion. It is induced by hyperosmolarity of glucose in the duodenum.

Ghrelin Shown to activate the endothelial form of nitric oxide synthase. Pro- duced by adipose tissue to induce satiation when present at high levels, it has been associated with obesity, insulin resistance and high blood pressure [Xu et al., 2008].

Glucagon Secreted by the pancreas, it raises blood glucose levels when they fall low by causing the liver to break down stored glycogen into glucose. GLP-1 Glucagon-like peptide-1, increases secretion of insulin, decreases

glucagon secretion, increases satiety in the brain, thus decreasing food intake and promotes insulin sensitivity of cells.

IL-6 Acts as both a pro and anti-inflammatory cytokine, secreted by T-cells and macrophages to stimulate the immune response after trauma. Also released from muscle cells, and is elevated in response to muscle contrac- tion. It has inhibitory e↵ects on TNF-↵, IL-1, IL-1ra and IL-10. Insulin Secreted by the pancreas in response to high blood glucose levels. It

stimulates cells to take up glucose from the blood and promotes the storage of glycogen. It inhibits the release of glucagon.

Leptin Plays a key role in energy uptake and energy expenditure, inducing ap- petite and metabolism. It promotes angiogenesis by increasing VEGF levels.

PAI-1 Plasminogen activator inhibitor-1, present in increased levels during obe- sity and metabolic syndrome.

Resistin Secreted by immune and epithelial cells, thought to play a role in in- flammation by increasing levels of IL-1, 6, 12 and TNF-↵. Thought to have links to both obesity and insulin resistance.

TNF-↵ Pro-inflammatory cytokine, involved in the regulation of immune cells, it can induce fever, induce apoptosis, sepsis (via IL-1 and IL-6 production), induce inflammation and inhibit viral replication and tumorigenesis. Visfatin Promotes vascular smooth muscle cell maturation, activates insulin re-

ceptors and has insulin-mimic e↵ects, such as lowering blood glucose levels and increasing insulin sensitivity.

Table 2.2: A list of metabolites related to diabetes, inflammation and metabolism, measured with a BioPlex machine.

and 2-plex assays. 25µl plasma samples were diluted by adding 75µl of human- specific BioPlex sample diluent. The samples were stored on ice until use, and 50µl of sample was used for the BioPlex assays.

A 96-well filter plate was pre-wet with 100µl of BioPlex assay bu↵er. The bu↵er was then removed by vacuum filtration, and the bottom of the filter plate was dried using a lint-free paper towel. The multiplex bead working solution was vortexed for 15-20 seconds at a medium speed, and 50µl was added to each well. The bu↵er was then removed by vacuum filtration, and the plate was washed twice with 100µl of BioPlex wash bu↵er, removing the bu↵er by vacuum filtration after each wash. 50µl of diluted sample or standard was added to each well, and the plate was covered with sealing tape. The plate was placed on a microplate shaker and covered with aluminium foil. The shaker speed was slowly increased to 1100rpm for 30 seconds, then reduced to 300rpm, and the plate was incubated at room temperature for 30 minutes.

After incubation, the sealing tape was removed and the bu↵er was removed by vacuum filtration. The sample was washed 3 times with 100µl of BioPlex wash bu↵er. The BioPlex detection antibody working solution was vortexed gently, and 25µl was added to each well. The plate was covered with a new piece of sealing tape and placed onto a microplate shaker, and covered with aluminium foil. The shaker speed was slowly increased to 1100rpm for 30 seconds, then reduced to 300rpm, and the plate was incubated at room temperature for 30 minutes.

After incubation, the sealing tape was taken o↵and the bu↵er was removed by vacuum filtration. The sample was washed 3 times with 100µl of BioPlex wash bu↵er. The 1 x streptavidin-PE was vortexed vigorously and 50µl was added to each well. The plate was covered with a new piece of sealing tape and placed onto a microplate shaker, and covered with aluminium foil. The shaker speed was slowly increased to 1100rpm for 30 seconds, then reduced to 300rpm, and the plate was incubated at room temperature for 10 minutes.

After incubation, the sealing tape was removed and the bu↵er was removed by vacuum filtration. The sample was washed 3 times with 100µl of BioPlex wash bu↵er. 125µl BioPlex assay bu↵er was added to each well to re-suspend the beads, and the plate was then covered in sealing tape. The plate was placed on a microplate shaker at 1100rpm for 30 seconds immediately before reading plate on the BioPlex system. The sealing tape was removed before reading.

The plate was placed in a Bio-Rad BioPlex 200 system, and read using the in- built software. Concentrations (pg/ml) of di↵erent cytokines in the plasma samples were determined by using the standard curves generated in the multiplex assays.

Each standard curve was generated using eight data points, and a nonlinear least squares minimisation algorithm was used for the curve fitting by the five-parameter logistic equation and to determine the high and low limits of detection.