Multiscreen harvest plates

Cell proliferation was measured by ³H-thymidine (³H-TdR) incorporation. CD3⁺ cells cultured in the absence or in the presence of MSC in a transwell system were pulsed with 0.5 μCi/well ³H-TdR (5 Ci/mmole specific activity; GE Healthcare Europe GmbH, Milan, Italy) for 8 hours. At the end of incubation, cells were harvested onto Multiscreen Harvest plates (Millipore, Billerica, MA, USA) using a 96-well plate-automated cell harvester (Tomtec, Handem, CT, USA). Scintillation liquid (Fisher Chemicals, Leicester, UK) was then added and ³H-TdR incorporation was measured by liquid scintillation spectroscopy using a beta-counter (Chameleon TM 425-104 Multilabel Counter -Bioscan, Washington, USA). The results expressed in counts per minute (kcpm, cpm × 1000) are given as the mean value of triplicate wells. In the same experiments, CD3⁺ cells cocultured as already described were also analyzed by flow cytometry for Ki67 intranuclear expression to identify KI67⁺ cycling T cells.

A ³H-thymidine incorporation assay was performed on MOPC315.BM cells, that is, mouse myeloma cells [24 (link)]. The cells were suspended in RPMI 1640 (Lonza, Verviers, Belgium) supplemented with 10% fetal bovine serum (Sigma-Aldrich, Diegem, Belgium) and 1% Penicillin/Streptomycin (Sigma-Aldrich), plated in 96-well plates (5 × 10⁴/100 μL/well) and cultured for 24 hours at 37°C and 5% CO₂ in the presence of different AndoSan concentrations. For the last 16 hours of culture, 0.17 μCi of [Methyl-³H] thymidine (Perkin Elmer, Zaventem, Belgium) was added to each well. DNA was harvested on Multiscreen Harvest Plates (Millipore, Carrightwohill, Cork, Ireland) using Filter Mate Harvester (Perkin Elmer). Plates were dried for 3-4 hours before adding 25 μL/well of Microscint O (Perkin Elmer) followed by radioactivity measurement (c.p.m.) with TopCount NXT Microplate Scintillation Counter (Perkin Elmer).

Spleens were harvested from C57BL/KaLwRij mice and cell suspensions were obtained as described above. Splenocytes (1×10⁵/well) were cultured in 96-well plates using complete RPMI 1640 medium containing 5 μM of 2-mercaptoethanol (Gibco by Life Technologies, Gent, Belgium). MDSC subsets were added (final culture volume 200 μl/well) at different MDSC:splenocyte ratios (1:8; 1:4; 1:2; 1:1) and T-cell proliferation was induced by adding 1 μl/well of mouse T-activator CD3/CD28 dynabeads (Gibco). Proliferation was assessed after 72 hours of culture by adding 0.33 μCi of [methyl-³H] thymidin (Perkin Elmer, Waltham, USA) to each well for the last 18 hours of culture. DNA was harvested on Multiscreen Harvest Plates (Millipore, Carrigtwohill, Ireland) using Filter Mate Harvester (Perkin Elmer). Plates were dried for 3-4 hours before adding 25 μl/well of Microscint-O (Perkin Elmer) and measuring radioactivity (c.p.m.) with a TopCount NXT Microplate Scintillation counter (Perkin Elmer). Suppression was calculated as follows: Suppression (%) = [1 − (Proliferation with MDSCs/ proliferation without MDSCs)] x 100.