Serum free medium

These methods and experimental protocols were carried out in accordance with the approved guidelines as approved by the National Research Ethics service and the University of Manchester ethics committee number 10/H1013/27. Written informed consent was obtained from all patients. Femoral heads were collected from patients undergoing total hip or knee replacement surgery and stored in serum-free DMEM with antibiotics/antimycotics until processed (Supplementary Table S1). Femoral head cartilage was shaved off using a scalpel and incubated overnight with agitation in serum-free DMEM containing an enzymatic cocktail of 0.25% collagenase type II (Life Technologies) and 0.1% hyaluronidase in serum-free medium (Sigma) to release the AC. The next day the solution was centrifuged and the pelleted ACs resuspended and plated in DMEM supplemented with 10% foetal calf serum (FCS) at a density of approximately 5,000–6,000 per cm². Cells were then incubated for 24–48 h in the above medium to allow cells to reach ∼80% confluency. The cells were then ‘serum starved’ for 24 h by replacing the normal, 10% FCS growth medium with medium containing 1% FCS (all other components unchanged). Cells were then treated as indicated in specific experiments.

Transwell permeable supports, 6.5 mm diameter inserts, 8.0 um pore size, polycarbonate membranes (Corning Inc.) were used to perform migration assay. Cells (10⁵) were seeded in the upper chamber of the transwell insert in serum-free medium (Sigma). The lower chamber of the transwell was filled with 600 uL of culture medium containing 10% of fetal bovine serum. Cells were incubated at 37°C and 5% CO₂ for 16 h, then transwells were removed and stained with 0.1% Crystal Violet (Sigma) in 25% methanol. Non-migrated cells were scraped off the top of the transwell with a cotton swab. Migrated cells were quantified by eluting Crystal Violet with 1% SDS and reading the absorbance at 550 nm using the microplate reader Infinite 200 (TECAN). The data showed the means and the s.e.m from one representative experiment out of three. The comparisons between two classes were performed by two-sample paired t-test.

At indicated time-points post 1^st and 2^nd doses of hNIs, vehicle-treated and hNI-treated NOD/SCID mice, and an additional group of age-matched, non-diabetic NOD/SCID control mice (n = 6) were fasted 5 hrs, whereupon baseline blood glucose levels were measured. Animals were anesthetized and 2 g glucose/kg b.wt. (dissolved in 0.5 ml serum free medium and filter sterilized; Sigma, St. Louis, MO) were administered via i.p. injection. Tail vein blood glucose levels were determined at 30 min, 60 min and 120 min post glucose administration. Human insulin levels in the sera of hNI and vehicle treated groups of mice were assayed by ELISA, following the manufacturer’s instructions (Mercodia, Uppsala, Sweden).

The positive clone hybridoma cells were cultured with serum-free medium (Sigma-Aldrich) to obtain their purified antibodies using HiTrap™ Protein G HP column chromatography (GE Healthcare, Uppsala, Sweden) according to the manufacturer’s protocol. The antibody subtypes were detected by the SBA Clonotyping System-HRP (Southern-Biotech, Birmingham, AL, USA) according to the manufacturer’s instructions.

Migration of CD8⁺ T cells was analyzed through the Transwell assay. CD8⁺ T cells (2 × 10⁴) isolated by microbeads from healthy donors were activated with CD3/CD28 beads and seeded in the upper chamber of the Transwell apparatus with serum-free medium (Millipore, Billerica, MA, USA). HeLa cells (2 × 10⁴) were seeded in the lower chamber with RPMI1640 medium. The number of CD8⁺ T cells was calculated using flow cytometry.