Round bottomed 96 well plate

The mixed lymphocyte reactions (MLR) consisted of 5 × 10⁴ murine CD4⁺ effector cells stimulated with anti-CD3 (1 μg/ml), anti-CD28 (1 μg/ml), IL-1β (20 ng/ml), IL-6 (20 ng/ml), and IL-23 (20 ng/ml) to induce Th17 cells and 5 × 10⁴ irradiated (40 Gy) allogeneic murine peripheral blood mononuclear cells (PBMCs) in round-bottomed 96-well plates (Nunc, Roskilde, Denmark) with PBMC culture medium (PCM) consisting of MEM-a supplemented with 2 mM L-glutamine, 1 % penicillin/streptomycin (P/S) and 10 % heat-inactivated human serum. Suppression assays were performed to determine the immunomodulatory capacities of Ad-hMSCs (various concentrations), Ad-hMSCs (1:10) and rapamycin treated-Ad-hMSCs (1:10) (alul ratios: indicated cells/effector cells) in the MLR. CD4⁺ T cell proliferation was investigated by adding ³H-thymidine (1 μCi/well; GE Healthcare, Amersham, Buckinghamshire, UK) to the culture medium and incubating for 8 h. The level of ³H-thymidine incorporation was measured using a Beckman liquid-scintillation counter (Beckman Coulter Inc., Brea, CA, USA).

DU145 and PC-3 PCa cell lines were cultured and harvested at 70–80% confluence according to standard laboratory protocols. Cell pellets were collected from DU145 and PC-3 cell lines to serve as controls from which total RNA was isolated. The remaining cells (5 × 10⁵) were re-suspended in sterile PBS containing 1% BSA. Using a MoFlo XDP high speed cell sorter (Beckman Coulter, USA), a single cell from both cell lines was seeded separately into each well, in triplicate, of 10 round-bottomed 96-well plates (Nunc, Germany) containing culture media. Two days following plating, the 96-well plates were examined using light microscopy and wells containing only one viable cell were identified. Seven days following plating, colonies derived from single cells were classified as holo-, mero-, and paraclones based on cell morphology^{12 (link)}. Images were acquired and holoclones were harvested at 14 days. At this time-point, wells containing holoclones were washed trypsinised and holoclones from all wells from each replicate were pooled together. Resulting pellets were stored for subsequent analysis. The media used in these experiments was the same as that used for standard culture of the cells.

Spleens from C57Bl/6 and CD1d^−/− mice were homogenized into single-cell suspensions and red blood cells removed using lysis buffer according to the manufacturer’s protocol (Sigma). Cells were then stimulated for up to 48 h with different concentrations of αGC before expression of CD69 on NK1.1⁺, TCRb-, B220- NK cells were assessed using flow cytometry and a FACSVerse (BDBiosciences). In some experiments, splenocytes were first labeled with antibodies against CD11c, NK1.1, TCRb and B220 (all BDBiosciences) before DCs (defined as TCRb-, B220-, NK1.1-, CD11c+), NK cells (defined as TCRb-, B220-, NK1.1⁺, CD11c-) and iNKT cells (defined as TCRb+, B220-, NK1.1⁺, CD11c-) were sorted out using a FACSAria II (BDBiosciences). Sorted cells were then co-cultured in round-bottomed 96-well plates (Nunc) using 25,000 WT NK cells, 10,000 WT iNKT cells and 10.000 DCs from C57Bl/6 WT or CD1d^−/− mice per well, where indicated, in the presence or absence of 1 ug/mL αGC for up to 48 h. The expression of CD69 was assessed by flow cytometry as described above.